Methods

ABSTRACT

The present invention provides method, uses and agents for preventing or reducing cognitive decline in a patient following a planned inflammatory trigger. Such planned inflammatory trigger can be a surgical procedure or chemotherapy. The invention further provides methods, uses and agents for reducing cognitive decline in a patient with a cognitive disorder, wherein said patient has been exposed to an inflammatory trigger. Pharmaceutical compositions and kits are also provided.

The present invention relates to methods, uses and agents for preventingor reducing cognitive decline in patients following surgery or otherinflammatory triggers.

Cognition, the mental activity involved in memory, attention andperception, may often decline as a result of illness (Forton et al,2001; Hopkins et al, 2005; Helfin et al, 2005). An impairment ofcognitive functions features prominently in major infective diseases(Capuron et al, 1999); the inflammatory response to infection,associated with elevated cytokines in the periphery, signals to thebrain to produce an array of symptoms ranging from lethargy to socialwithdrawal and memory impairment, collectively known as sicknessbehaviour (Dantzer, 2004). The function of sickness behavior is topromote recovery from illness and injury (Bolles and Fanselow, 1980).

An impairment of cognition has also been shown to develop as aconsequence of surgery (Moller et al, 1998). Termed post-operativecognitive dysfunction (POCD), it features disturbance of memory,attention, consciousness, information processing and sleep-wake cycle,leading to postoperative morbidity and mortality (Bohnen et al, 1994;Monk et al, 2008). The highest incidence of POCD occurs in elderlypatients (Moller et al, 1998), but other age groups are also affected(Johnson et al, 2002).

The precise pathogenesis of POCD is not known and may involveperioperative as well as patient-related factors (Newman et al, 2007);general anesthetics have been implicated, as animal studies suggest thatanesthetic-induced changes in the brain outlast the elimination ofanaesthetic agents from the body (Fütterer et al, 2004) and are capableof producing long-lasting cognitive dysfunction under certaincircumstances (Culley et al, 2003). Yet there appears to be no decreasein the incidence of POCD after regional anesthesia (Campbell et al,1993; Williams-Russo et al, 1995); therefore, a causative role forgeneral anesthetics appears to be unlikely.

Wan et al (2007) Anaesthesiology 106: 436-43 suggested that cognitivedecline following splenectomy in adult rats is associated with ahippocampal inflammatory response that may be due to pro-inflammatorycytokine-dependent activation of glial cells.

Rosczyk et al (2008) Exp. Gerontology 43: 840-46 found that minorsurgery leads to an exaggerated neuroinflammatory response in aged mice(compared to young mice) but that this did not result in significantlyimpaired performance in a memory test.

Using in vivo models of cognitive function, the present inventors havesurprisingly found that ablation of interleukin-1 (IL-1) signallingand/or Tumour Necrosis Factor α (TNFα) signalling in vivo preventedsurgery-induced cognitive decline.

In a first aspect, the present invention provides a method forpreventing or reducing cognitive decline in a patient following aplanned inflammatory trigger in said patient, the method comprisingadministering a therapeutically effective amount of a Tumour NecrosisFactor alpha (TNFα) antagonist to said patient.

In a second aspect, the present invention provides for the use of atherapeutically effective amount of a Tumour Necrosis Factor alpha(TNFα) antagonist in the manufacture of a medicament for use inpreventing or reducing cognitive decline in a patient following aplanned inflammatory trigger in said patient.

In a third aspect, the present invention provides an agent for use inpreventing or reducing cognitive decline in a patient following aplanned inflammatory trigger in said patient, wherein the agentcomprises a therapeutically effective amount of a Tumour Necrosis Factoralpha (TNFα) antagonist.

By a “planned inflammatory trigger” we include the meaning of a plannedmedical procedure that may be expected to lead to an inflammatoryresponse in the patient, and where the planned inflammatory trigger hasbeen associated with cognitive decline in patients. Thus, this may beany procedure that has been associated with post-procedure impairedcognition, that may be for example, delirium, dementia, confusion, asdefined below.

In an embodiment of the preceding aspects, the planned inflammatorytrigger is surgery and the method, use or agent is thus for preventingor reducing post-operative cognitive dysfunction (POCD) in said patient.

In an alternative embodiment of the preceding aspects, the plannedinflammatory trigger is chemotherapy. Delirium and other symptoms ofcognitive disorders have been associated with chemotherapy treatment ofcancer patients. Thus, it is envisaged that the present invention may beutilised to prevent or reduce cognitive decline in cancer patientsfollowing chemotherapy treatment, either prophylactically, or as atreatment, as described below.

By “cognitive decline” we include the meaning of any deterioration ofcognitive function brought about by a cognitive disorder and/or aninflammatory trigger as defined below.

By “post-operative cognitive dysfunction”, we include the deteriorationof intellectual function reflected as memory and concentrationimpairment presenting in a patient after that patient has undergone asurgical procedure. Such deterioration of intellectual function may takemany forms and as such this definition includes any form of cognitivedecline presenting post-operatively. The present invention is consideredto be particularly useful when administered before, during orimmediately following surgery. In general, cognitive dysfunctionsfollowing surgery are common and effective immediately followingrecovery. Classical POCD characterises a more prolonged and subtledysfunction in cognitive domains, juxtaposed to a more evident butshort-lived “delirium” (both are included in the above definition ofPOCD). Discrimination between cognitive dysfunctions is made inparticular according to the length of the cognitive impairment; deliriumresolves itself usually after few days, whereas POCD persists for months(>3) and can become a permanent dysfunction. Thus, such cognitivedecline falling within the scope of the above definition may beshort-lived, thus may ablate hours or days after completion of thesurgical procedure; or the cognitive decline may persist over the courseof months or years, or the cognitive decline may even be permanent.Delirium is commonly seen after surgery, usually soon after surgery(hours to days) and fluctuating over time. Although the dysfunctionlasts over a short period of time, delirium is associated with increasedmortality (Ely et al. 2004), greater care dependency, costs (Milbrandtet al. 2004) and prolonged hospitalization (Ely et al. 2001). It isconsidered that the use of the present invention will aid in reducing orpreventing this deterioration of intellectual function and lead to animprovement in the quality of life of the patient and his/her carers.

The diagnosis of POCD may be aided by neuropsychological testing. Ingeneral, the presence of POCD may be suspected when memory loss isgreater than expected under normal situations. At present, there are nospecific cognitive sets for successful POCD diagnosis; generallymultiple neurocognitive assessments are made before reaching a diagnosis(Newman S et al, Anesthesiology 2007, 106(3): 572-90).

It is envisaged that the symptoms of POCD may include memory loss,memory impairment, concentration impairment, delirium, dementia, and/orsickness behaviour.

By “delirium” is included an acute and debilitating decline inattention, focus, perception, and cognition that produces an alteredform of semi-consciousness. Delirium is a syndrome, or group ofsymptoms, caused by a disturbance in the normal functioning of thebrain. The delirious patient has a reduced awareness of andresponsiveness to the environment, which may be manifested asdisorientation, incoherence, and memory disturbance. Delirium affects atleast one in 10 hospitalised patients, and 1 in 2 elderly hospitalisedpatients. Whilst it is not a specific disease itself, patients withdelirium usually fare worse than those with the same illness who do nothave delirium. It occurs as a post-operative complication, with evidencefrom the mouse model described in the Examples showing that it can becaused by an inflammatory trigger. This would also explain why deliriumis seen in patients admitted to hospital as a result of otherinflammatory triggers, for example, stroke (CVA), Heart Attack (MI),urinary tract infection (UTI), respiratory tract infection (RTI),poisoning, alcohol or other medication withdrawal, hypoxia, and headinjury.

By “dementia” we mean a serious cognitive disorder, which may be static,the result of a unique global brain injury or progressive, resulting inlong-term decline in cognitive function due to damage or disease in thebody beyond what might be expected from normal aging.

By “sickness behaviour” are included symptoms ranging from lethargy,fever, decreased food intake, somnolence, hyperalgesia, and generalfatigue to social withdrawal and memory impairment (Dantzer R:Cytokine-induced sickness behaviour: a neuroimmune response toactivation of innate immunity. Eur J Pharmacol 2004, 500(1-3):399-411).

The present inventors have demonstrated that microglial activation andassociated inflammation are associated with the onset of POCD. Further,the ablation of microglial activation with minocycline was found toprevent post-operative memory loss in the in vivo models used. Thus, amethod for assessing the onset of, or the progress of treatment for,POCD may be the analysis of microglial activation in the brain of thepatient. Such activation may be measured using techniques such asPositron Emission Tomography (PET) scanning of the patient's brain. SuchPET scanning may, for example, be conducted using ¹¹C-PK11195, which isa ligand for the peripheral benzodiazepine receptor. An elevation ofmicroglial activation may be an indication of POCD (or vice versa).Further methods of assessing POCD may include magnetic resonance imaging(MRI) or PET with FEPPA or ¹¹C-PK11195. Other imaging techniquesincluding MRI with diffusion tensor imaging and MR spectroscopy can alsobe used to non-invasively assess POCD.

Risk factors for the development of POCD include advanced age in thepatient, the patient's level of education and “cognitive reserve”,potential genetic polymorphisms (for example APOe4) and co-morbidities,such as underlying neurological disease.

By “preventing POCD” we include the meaning that the method, use oragent of the invention is considered to reduce the likelihood of theoccurrence of POCD in a patient who has undergone a surgical procedure.Thus, the invention may be used, or be for use, prophylactically beforeany sign of POCD develops in the patient. While it is preferred thatPOCD is prevented from occurring in the patient, it is understood thatsome incidence of POCD may still remain but it is envisaged that the useof the present invention will reduce the symptoms of, and/or reduce thepersistence of, that POCD. Thus, by “reducing POCD” we include themeaning that the onset of POCD is lessened or delayed and the symptomsare reduced thus improving the cognition of the patient while perhapsnot entirely preventing the onset of the POCD. This can be establishedby a battery of neuropsychological tests. The invention may also be usedfollowing presentation of POCD in a patient, as a treatment for thePOCD.

A further aspect of the invention provides a method for reducingcognitive decline in a patient with a cognitive disorder, wherein saidpatient has been exposed to an inflammatory trigger, the methodcomprising administering a therapeutically effective amount of a TumourNecrosis Factor alpha (TNFα) antagonist to said patient after exposureof said patient to said inflammatory trigger.

Thus, a further aspect of the invention provides for the use of atherapeutically effective amount of a Tumour Necrosis Factor alpha(TNFα) antagonist in the manufacture of a medicament for use in reducingcognitive decline in a patient with a cognitive disorder, wherein saidpatient has been exposed to an inflammatory trigger.

A yet further aspect of the invention provides an agent for use inreducing cognitive decline in a patient with a cognitive disorder,wherein said patient has been exposed to an inflammatory trigger, andwherein the agent comprises a therapeutically effective amount of aTumour Necrosis Factor alpha (TNFα) antagonist.

By “cognitive disorder” we include the meaning of any neurologicaldisease, condition or disorder that manifests in impaired cognitivefunction in a patient. Such disorders may arise in patients of any age.The symptoms of such disorders may include drowsiness, fatigue,concentration impairment, vertigo, confusion, memory impairment, memoryloss, delirium, loss of motor neurone control and other such symptoms aswould be understood by a person of skill in the art. As explained above,delirium is a symptom, or group of symptoms, but is also a syndrome,which is caused by a disturbance in the normal functioning of the brain.Thus, it is envisaged that while delirium may be a symptom of thecognitive disorder, where another disorder is present, it may also bethe only cognitive disorder that has presented in the patient and thusthe present invention may be beneficial where no other cognitivedisorder has yet been characterised but the patient is exhibiting signsof delirium. Further examples of cognitive disorders encompassed hereininclude Alzheimer's Disease, multiple sclerosis, stroke, Parkinson'sDisease, Huntington's Disease, dementia, frontotemporal dementia,vascular dementia, HIV dementia, Post-Traumatic Stress Disorder andchronic inflammatory conditions such as Rheumatoid Arthritis. Furtherexamples of relevant conditions would be known to the skilled person.

By “reducing cognitive decline” we include the meaning that theprogression of the symptoms of the cognitive disorder over time isslowed and those symptoms are lessened and potentially reversed by theuse of the invention. The invention may be used to improve cognitivefunction by reducing the onset of cognitive decline. It is hoped thatsuch improvement may provide patients with greater independence and agreater quality of life.

By “inflammatory trigger” we include the meaning of any insult to thebody that results in an inflammatory response. Such an inflammatoryresponse, if left unchecked, may lead to an overactive neuroinflammatoryresponse and cause or worsen (if a cognitive disorder is alreadypresent) the cognitive condition of patients. A non-exhaustive list ofexamples of such inflammatory triggers includes infection, trauma (suchas broken bones after a fall), surgery, vaccination, arthritis, obesity,diabetes, stroke (CVA), cardiac arrest (heart attack; myocardialinfarction (MI)), burns, chemotherapy, blast injury, urinary tractinfection (UTI), respiratory tract infection (RTI), Humanimmunodeficiency virus infection (HIV), poisoning, alcohol or othermedication withdrawal, hypoxia, and head injury.

It is envisaged that the patient, while potentially not already havingbeen diagnosed with a cognitive disorder, may be at risk of developing acognitive disorder. Thus, the present invention may also be beneficialto patients who are at risk of developing a cognitive disorder. Suchpatients may include the elderly or individuals who have a familialhistory of such disorders.

In an embodiment of the methods of the present invention, the methodsmay further comprise administering a therapeutically effective amount ofan Interleukin 1 (IL-1) antagonist to said patient.

Thus, in an embodiment of the uses and agents of the present invention,the medicament or agent may be for administration in combination with atherapeutically effective amount of an Interleukin 1 (IL-1) antagonist.

It is envisaged that in the preceding embodiments of the invention, theIL-1 antagonist may be administered, or may be for administration,before, after or simultaneously with the TNFα antagonist. Thus, theefficacy of the invention may be improved by administering the IL-1antagonist and the TNFα antagonist at time points where their individualefficacies may be greatest. Thus, it is envisaged that the IL-1antagonist and the TNFα antagonist may be formulated separately.

In an alternative embodiment, the IL-1 antagonist may be co-formulatedwith the TNFα antagonist. Thus, in this embodiment, the administrationof the IL-1 antagonist and the TNFα antagonist will be simultaneous.

In a further alternative, the IL-1 antagonist and the TNFα antagonistmay be comprised in a single molecule, such as a chimeric molecule, forexample, a fusion protein. Thus, a compound with both IL-1 antagonistactivity and TNFα antagonist activity may be used in the methods anduses of the invention. Such compound may comprise, for example, a fusionof antigen-binding regions of antibodies with IL-1 antagonist activityand TNFα antagonist activity.

In an embodiment of any aspect of the present invention the antagonist(either IL-1 antagonist, TNFα antagonist or both) may be administered(in the methods of the invention), or may be for administration (in theuses and agents of the invention) systemically. Such systemicadministration may, for example, be by intravenous (i.v.) administrationin an appropriate formulation. It is envisaged that i.v. administrationwill lead to a rapid and more efficacious effect. An example of anembodiment where systemic administration may be appropriate includesadministration to patients who are undergoing multiple surgicalprocedures, or where the surgical procedure results in major trauma tothe body.

It is considered that the antagonists of the invention will actperipherally, but in some circumstances the agents may cross theblood:brain barrier to act directly on the brain and the central nervoussystem.

It is envisaged that the antagonist may be formulated as appropriate forthe type of surgical procedure or cognitive disorder in question.Appropriate formulations will be evident to a person of skill in the artand may include, but are not limited to, the group comprising a liquidfor injection or otherwise, an infusion, a cream, a lozenge, a gel, alotion or a paste. The antagonist of the invention may also be foradministration in biocompatible organic or inorganic matrices including,but not limited to, collagen or fibronectin matrices. It is envisagedthat such matrices may act as carriers of the antagonist in anappropriate formulation or may aid in the reduction of inflammation byaugmenting the effects of the antagonist.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Such methods include the step of bringing into association the activeingredient (antagonist of the invention) with the carrier whichconstitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

In human or animal therapy, the antagonist of the invention can beadministered alone but will generally be administered in admixture witha suitable pharmaceutical excipient, diluent or carrier selected withregard to the intended route of administration and standardpharmaceutical practice.

The antagonists of the invention can be administered parenterally, forexample, intravenously, intra-arterially, intraperitoneally,intrathecally, intraventricularly, intrasternally, intracranially,intra-muscularly or subcutaneously, or they may be administered byinfusion techniques. They may be best used in the form of a sterileaqueous solution which may contain other substances, for example, enoughsalts or glucose to make the solution isotonic with blood. The aqueoussolutions should be suitably buffered (preferably to a pH of from 3 to9), if necessary. The preparation of suitable parenteral formulationsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well-known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose or an appropriate fraction thereof, of an activeingredient. It is preferred that doses for topical administration of theantagonists of the invention may be of the order of fractions of ormultiple mg/kg body weight of the patient. For example, the dose may bebetween 0.01 to 500 mg/kg body weight; 1 to 400 mg/kg body weight; 2 to200 mg/kg body weight; 3 to 100 mg/kg body weight or 4 to 50 mg/kg (orany combination of these upper and lower limits, as would be appreciatedby the skilled person). The dose used may in practice be limited by thesolubility of the compound. Examples of possible doses are 0.01, 0.05,0.075, 0.1, 0.2, 0.5, 0.7, 1, 2, 5, 10, 12, 15, 20, 25, 30, 35, 40, 45,50 or 100 mg per kg body weight up to, for example 500 mg/kg bodyweight, or any value in between. It is envisaged that preferred doses ofantagonist would be adjusted according to relative potency. Thephysician or veterinary practitioner will be able to determine therequired dose in a given situation based on the teaching and Examplesprovided herein.

Alternatively, the antagonists of the invention may be applied topicallyin the form of a lotion, solution, cream, ointment or dusting powder.The antagonists of the invention may also be transdermally administered,for example, by the use of a skin patch.

For application topically to the skin, the antagonists of the inventioncan be formulated as a suitable ointment containing the active compoundsuspended or dissolved in, for example, a mixture with one or more ofthe following: mineral oil, liquid petrolatum, white petrolatum,propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifyingwax and water. Alternatively, they can be formulated as a suitablelotion or cream, suspended or dissolved in, for example, a mixture ofone or more of the following: mineral oil, sorbitan monostearate, apolyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, water and dimethylsulphoxide (DMSO).

Formulations suitable for topical administration in the mouth (such asin the dental embodiments of the invention) include lozenges comprisingthe active ingredient in a flavoured basis, usually sucrose and acaciaor tragacanth; pastilles comprising the active ingredient in an inertbasis such as gelatin and glycerin, or sucrose and acacia; andmouth-washes comprising the active ingredient in a suitable liquidcarrier.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

For veterinary use, a compound of the invention is administered as asuitably acceptable formulation in accordance with normal veterinarypractice and the veterinary surgeon will determine the dosing regimenand route of administration which will be most appropriate for aparticular animal.

In embodiments of the invention relating to preventing or reducingcognitive decline following a planned inflammatory trigger, the TNFαantagonist may be administered, or may be for administration, before,during or after the planned inflammatory trigger. For example, the TNFαantagonist may be administered, or may be for administration, before thecommencement of a surgical procedure on said patient. Suchadministration may be immediately before surgery or several seconds,minutes or even hours before surgery. Alternatively, the TNFα antagonistmay be administered, or may be for administration, during a surgicalprocedure on said patient.

In yet another alternative, the TNFα antagonist may be administered, ormay be for administration, after completion of a surgical procedure onsaid patient. In this embodiment, it is envisaged that the TNFαantagonist may be administered, or may be for administration, up to 1hour after completion of said surgical procedure. Alternatively, theTNFα antagonist may be administered, or may be for administration,between 0 seconds (i.e. immediately after completion of the surgicalprocedure) up to 1 day after completion of the surgical procedure. Thusthis may be between 5 seconds and 10 hours after completion of thesurgical procedure. Preferably, this may be between 30 seconds and 1hour, for example 30 minutes, after completion of the surgicalprocedure. Such administration (or administration in any other aspect ofthe invention) may comprise a single administration of a single dose, ormay comprise multiple administrations of the same, increasing, ordecreasing doses, as appropriate. Thus, the administration may be over acourse of time as prescribed by the physician.

Accordingly, the TNFα antagonist may be administered, or may be foradministration to the patient; before commencement of chemotherapy;during chemotherapy; or after completion of a round of treatment ofchemotherapy on said patient, when the planned inflammatory trigger ischemotherapy. The administration regimes described herein in relation tosurgery also apply equally to other planned inflammatory triggers,including chemotherapy.

It is envisaged that patients who may benefit from the aspects of theinvention relating to POCD may have, or be at risk of developing,delirium, Alzheimer's Disease, multiple sclerosis, stroke, Parkinson'sDisease, Huntington's Disease, dementia, frontotemporal dementia,vascular dementia, HIV dementia, Post-Traumatic Stress Disorder orchronic inflammatory conditions such as Rheumatoid Arthritis.

In any aspect of the invention the patient may be a mammal. Such mammalmay be a domestic pet (such as a dog or cat), a farm animal (such as acow, pig, sheep, goat or buffalo), a sport animal (such as a horse) or alaboratory animal (such as a mouse, rat, rabbit, guinea pig, gerbil,hamster, monkey or ape). It is preferred that the patient is a human.

When the patient is a human, it is envisaged that they may be often over50 years of age. Advanced age is a risk factor for cognitive decline andPOCD thus it is envisaged that older patients may benefit more from thepresent invention than younger individuals. However, in a furtheraspect, the patient may be less than 20 years of age. Children who areborn with genetic abnormalities, congenital conditions or who for anyreason require surgical treatments early in life may be at risk ofdeveloping POCD and other cognitive conditions. Thus, it is alsoenvisaged that the present invention may benefit younger patients.Nevertheless, the present invention will be useful to patients of anyage who have to undergo surgical procedures for any reason and/or whomay be at risk of developing a cognitive disorder.

By “TNFα antagonist” we include the meaning that the antagonist is anycompound that antagonises, thus decreases or ablates, the effects ofTNFα. Thus the antagonist may be a compound that targets TNFα itself, acompound that targets any upstream effector of TNFα or a compound thattargets any downstream effector of TNFα. By “targeting TNFα itself” wemean blocking or reducing the transcription, translation,post-translational modification of precursors, or release of TNFα fromcells where it is synthesised, as would be understood by a person ofskill in the art. By “targets any upstream effector of TNFα” we mean anysignal or molecule that triggers the synthesis and/or release of TNFα.By “targets any downstream effector of TNFα” we mean any receptor orother compound that interacts with TNFα to bring about its effects invivo.

Thus, in any aspect of the invention, the TNFα antagonist may be a TNFαreceptor antagonist.

The TNFα antagonist may be an antibody, an antibody fragment or fusionthereof. Thus, the TNFα antagonist may be an anti-TNFα antibody orfragment or fusion thereof. Such antibodies may be polyclonal ormonoclonal. Non-human antibodies may be humanised, for use in humanpatients. The antibodies may alternatively be chimeric, as would beunderstood by a person of skill in the art. The antibody fragment may bea Fab, Fv, ScFv or dAb, as would be understood by those skilled in theart. By “ScFv molecules” we mean molecules wherein the V_(H) and V_(L)partner domains of the antibody are linked via a flexible oligopeptide.

The advantages of using antibody fragments, rather than wholeantibodies, are several-fold. The smaller size of the fragments may leadto improved pharmacological properties, such as better penetration ofsolid tissue. Effector functions of whole antibodies, such as complementbinding, are removed. Fab, Fv, ScFv and dAb antibody fragments can allbe expressed in and secreted from E. coli, thus allowing the facileproduction of large amounts of the said fragments. Whole antibodies, andF(ab′)₂ fragments are “bivalent”. By “bivalent” we mean that the saidantibodies and F(ab′)₂ fragments have two antigen combining sites. Incontrast, Fab, Fv, ScFv and dAb fragments are monovalent, having onlyone antigen combining site.

Many available TNFα antagonists may be useful in the context of thepresent invention. Agents which bind TNFα and block its action includeanti human TNFα monoclonal antibodies, marketed examples includeinfliximab (Remicade®), adalimumab (Humira®), human TNF-R fusion proteinsuch as etanercept (Enbrel®) or other agents which resemble antibodieswhich bind TNF.

Inhibitors of TNF receptor include antibodies or antibody-like molecules(fragments, chains, dAbs etc) which bind to TNF receptors, more aremarketed at present.

Inhibitors of TNF signalling, and signalling pathways, includeinhibitors of NFκB. MAP kinases etc. could also be used.

Alternatively, the TNFα antagonist may be a small chemical entity. Suchchemical entities may be identified through high-throughput screening ofcompound libraries, or they may be designed in silico to interact withtheir intended target, such as receptors for TNFα.

In an alternative embodiment, the TNFα antagonist may be an siRNAmolecule, an antisense oligonucleotide or a ribozyme. Thus, suchantagonists may inhibit the transcription and/or translation of TNFα, asappropriate. Antisense oligonucleotides are single-stranded nucleicacids, which can specifically bind to a complementary nucleic acidsequence. By binding to the appropriate target sequence, an RNA-RNA, aDNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are oftentermed “antisense” because they are complementary to the sense or codingstrand of the gene. Recently, formation of a triple helix has provenpossible where the oligonucleotide is bound to a DNA duplex. It wasfound that oligonucleotides could recognise sequences in the majorgroove of the DNA double helix. A triple helix was formed thereby. Thissuggests that it is possible to synthesise sequence-specific moleculeswhich specifically bind double-stranded DNA via recognition of majorgroove hydrogen binding sites.

By binding to the target nucleic acid, the above oligonucleotides caninhibit the function of the target nucleic acid. This could, forexample, be a result of blocking the transcription, processing,poly(A)addition, replication, translation, or promoting inhibitorymechanisms of the cells, such as promoting RNA degradations.

Antisense oligonucleotides are prepared in the laboratory and thenintroduced into cells, for example by microinjection or uptake from thecell culture medium into the cells, or they are expressed in cells aftertransfection with plasmids or retroviruses or other vectors carrying anantisense gene.

Typically, antisense oligonucleotides are 15 to 35 bases in length. Forexample, 20-mer oligonucleotides have been shown to inhibit theexpression of the epidermal growth factor receptor mRNA (Witters et al,Breast Cancer Res Treat 53:41-50 (1999)) and 25-mer oligonucleotideshave been shown to decrease the expression of adrenocorticotropichormone by greater than 90% (Frankel et al, J Neurosurg 91:261-7(1999)). However, it is appreciated that it may be desirable to useoligonucleotides with lengths outside this range, for example 10, 11,12, 13, or 14 bases, or 36, 37, 38, 39 or 40 bases.

Similarly, (cf TNFα antagonist) by “IL-1 antagonist” we include themeaning that the antagonist is any compound that antagonises, thusdecreases or ablates, the effects of IL-1. Thus the antagonist may be acompound that targets IL-1 itself, a compound that targets any upstreameffector of IL-1 or a compound that targets any downstream effector ofIL-1.

Thus, in any aspect of the present invention, the IL-1 antagonist may bean IL-1 receptor antagonist, an IL-1α antagonist, an IL-1β antagonist ora Toll-like receptor (TLR) antagonist. The IL-1 antagonist may be anantibody, an antibody fragment or fusion thereof. Thus, for example, theIL-1 antagonist may be an anti-IL-1β antibody. Alternatively, the IL-1antagonist may be a small chemical entity. In a further alternative, theIL-1 antagonist may bean siRNA molecule, an antisense oligonucleotide ora ribozyme. In one embodiment, the IL-1 receptor antagonist is anakinra(Kineret®).

In embodiments of the invention relating to preventing or reducingcognitive decline following a planned inflammatory trigger, the IL-1antagonist may be administered, or may be for administration, before,during or after the planned inflammatory trigger. For example, the IL-1antagonist may be administered, or may be for administration, before thecommencement of a surgical procedure on said patient. Suchadministration may be immediately before surgery or several seconds,minutes or even hours before surgery. Alternatively, the IL-1 antagonistmay be administered, or may be for administration, during a surgicalprocedure on said patient.

In yet a further alternative, the IL-1 antagonist may be administered,or may be for administration, to the patient after completion of asurgical procedure on said patient. In this embodiment, it is envisagedthat the IL-1 antagonist may be administered, or may be foradministration, up to 1 hour after completion of said surgicalprocedure. Alternatively, the IL-1 antagonist may be administered, ormay be for administration, between 0 seconds (i.e. immediately aftercompletion of the surgical procedure) up to 2 days after completion ofthe surgical procedure. Thus this may be between 5 seconds and 1 dayafter completion of the surgical procedure. Preferably, this may bebetween 30 seconds and 10 hours, for example 2 hours, after completionof the surgical procedure. Such administration (or administration in anyother aspect of the invention) may comprise a single administration of asingle dose, or may comprise multiple administrations of the same,increasing, or decreasing doses, as appropriate. Thus, theadministration may be over a course of time as prescribed by thephysician.

Accordingly, the IL-1 antagonist may be administered, or may be foradministration to the patient; before commencement of chemotherapy;during chemotherapy; or after completion of a round of treatment ofchemotherapy on said patient, when the planned inflammatory trigger ischemotherapy. The administration regimes described herein in relation tosurgery also apply equally to other planned inflammatory triggers,including chemotherapy.

It is envisaged that the surgical procedure in any aspect of theinvention, may be a cardiothoracic, an orthopaedic, a neurological, avascular, a plastic & reconstructive, a gynaecological, an obstetric, aurological, a general, a head & neck, an ear, nose & throat (ENT), apaediatric, a dental, a maxillofacial, an ophthalmic, a pain management,a trauma, or a minor surgical procedure. Examples of such generalsurgical procedure are colorectal, hepatobiliary, or uppergastro-intestinal surgical procedures. Examples of such minor surgicalprocedures are catheterisation, minor skin procedures, minor orthopedicprocedures, nerve blocks, endoscopies, transoesophageal echocardiogramsor other minor procedures.

It is envisaged that the surgical procedure may be carried out undergeneral anaesthesia, regional anaesthesia, local anaesthesia, sedationor a combination thereof. By “general anaesthesia” is meant anaesthesiawhere the patient is “asleep”, i.e. not conscious, during the surgicalprocedure. There are three phases of general anaesthesia: induction(getting off to sleep); maintenance (keeping asleep while having thesurgical procedure); and emergence (waking up after the operation).Different drugs are utilised at these different stages. It is envisagedthat the present invention may be suitable for application during allthree phases. It is envisaged that the antagonists of the presentinvention may be combined with other drugs, such as anaesthetic drugs,for ease of administration during the different phases of anaesthesia,if appropriate, as would be directed by the physician.

Regional anaesthesia involves an infusion or single injection of localanaesthetic sometimes with additives (opiods, clonidine, etc) at a siteaway from the operative field. For example, spinal (intrathecal)anaesthesia (caesareans, prostate surgery, knee surgery), epiduralanaesthesia, caudal anaesthesia, regional nerve blocks (Bier's block forthe arm). The present invention may also be useful for application insurgical procedures carried out under regional anaesthesia. Antagonistsof the present invention may be combined with anaesthetic formulationsfor ease of administration, if appropriate.

Local anaesthesia involves injection of local anaesthetic drugs close tothe area where the procedure is to be carried out.

An aspect of the present invention provides a kit of parts comprising anIL-1 antagonist and a TNFα antagonist for use in preventing or reducingcognitive decline in a patient following a planned inflammatory trigger.Such planned inflammatory trigger may be a surgical procedure and thekit may therefore be for preventing or reducing post-operative cognitivedysfunction (POCD) in a patient. Alternatively, the planned inflammatorytrigger may be chemotherapy. It is preferred that the IL-1 antagonist isan IL-1 receptor antagonist and the TNFα antagonist is an anti-TNFαantibody.

A yet further aspect of the invention provides a kit of parts comprisingan IL-1 antagonist and a TNFα antagonist for use in reducing cognitivedecline in a patient with a cognitive disorder, wherein said patient hasbeen exposed to an inflammatory trigger. It is preferred that the IL-1antagonist is an IL-1 receptor antagonist and the TNFα antagonist is ananti-TNFα antibody. The cognitive disorder may be as defined above.

The invention further provides a kit of parts comprising: an IL-1antagoinist; a TNFα antagonist; and instructions for administration ofsaid IL-1 antagoinist and TNFα antagonist to a patient before, during orafter a planned inflammatory trigger. Such planned inflammatory triggermay be a surgical procedure or chemotherapy, for example.

A further aspect of the present invention provides a method forpreventing or reducing cognitive decline in a patient following aplanned inflammatory trigger in said patient, the method comprisingadministering a therapeutically effective amount of an Interleukin 1(IL-1) antagonist to said patient. The planned inflammatory trigger maybe surgery and the method may therefore be for preventing or reducingpost-operative cognitive dysfunction (POCD). Alternatively, the plannedinflammatory trigger may be chemotherapy.

The IL-1 antagonist may be administered to the patient beforecommencement of a surgical procedure on said patient. Alternatively, theIL-1 antagonist may be administered to the patient during a surgicalprocedure on said patient. In a further alternative, the IL-1 antagonistmay be administered to the patient after completion of a surgicalprocedure on said patient. Such treatment regime applies accordinglywhen the planned inflammatory trigger is other than surgery, for examplechemotherapy.

A further aspect provides a method for reducing cognitive decline in apatient with a cognitive disorder, wherein said patient has been exposedto an inflammatory trigger, the method comprising administering atherapeutically effective amount of an Interleukin 1 (IL-1) antagonistto said patient after exposure of said patient to said inflammatorytrigger. It is envisaged that the cognitive disorder may be selectedfrom, but not limited to, the group comprising delirium, Alzheimer'sDisease, multiple sclerosis, stroke, Parkinson's Disease, Huntington'sDisease, dementia, frontotemporal dementia, vascular dementia, HIVdementia, Post-Traumatic Stress Disorder or chronic inflammatorydisorders such as Rheumatoid Arthritis.

The present invention further provides an antagonist of the presentinvention in combination with a pharmaceutically acceptable carrier.

All documents referred to herein are incorporated herein, in theirentirety, by reference.

The listing or discussion of a prior-published document in thisspecification should not necessarily be taken as an acknowledgment thatthe document is part of the state of the art or is common generalknowledge.

The invention is now described in more detail by reference to thefollowing, non-limiting, Figures and Examples.

FIGURES

FIG. 1. Surgery-induced systemic inflammation is associated withincreased expression of hippocampal IL-1β and is blocked by minocyclineIL-1β and IL-6 levels in plasma were measured by ELISA at 2, 6, 24 or 72hours post-intervention. Surgery resulted in increased plasma levels ofIL-1β (A) and IL-6 (B) compared to mice receiving the same anestheticswithout surgery (Anesthesia) or to naïve animals. Administration ofminocycline (40 mg/kg, i.p.), an antibiotic with anti-inflammatoryproperties, mitigated surgery-induced elevations in IL-1β and IL-6 inplasma. Enrofloxacin, a comparable antimicrobial to minocycline butdevoid of any anti-inflammatory properties failed to reduce plasmalevels of IL-1β, compared to surgical littermates (Surgery) injectedwith saline (n=6). Six hours after surgery IL-1β expression in thehippocampus was increased compared to naïve and anesthesia groups (C).Administration of minocycline but not enrofloxacin mitigatedsurgery-induced, IL-1β-mediated, hippocampal inflammation (n=7). Dataare expressed as mean ±SEM, ***p<0.001; *p<0.05; for comparison betweensurgery or enrofloxacin vs naïve, anesthetics and minocycline groups.

FIG. 2. Surgery induces transcription of IL-1β and IL-6 in thehippocampus. IL-1β (A) and IL-6 (B) mRNA were measured by quantitativereal time PCR (qRT-PCR) in hippocampal samples extracted 4, 6 or 24hours after surgery. Naïve animals were used as controls. Surgeryresulted in increased transcription of both IL-1β and IL-6 in thehippocampus compared to naïve group, 6 hours after surgery and hadreturned to normal by 24 hours after surgery (n=6). Data are expressedas mean fold change ±SEM, ***p<0.001; for comparison between surgery vsnaïve group.

FIG. 3. Immunohistochemistry of microglia with anti-CD11b (A, B, C, D).Hippocampi were harvested 1, 3 or 7 days after treatment (pictures shownrefer to tissue harvesting after 1 day) and stained with avidin-biotintechnique. Representative photomicrographs from naïve (A) anaestheticsalone (B) surgical (C), and surgical animals treated with minocycline(D). The amoeboid hypertrophy of cell bodies, as well as clumping ofprocesses seen following surgery is prevented by administration ofminocycline. Scale bar 30 μm. Median (horizontal bar) with 25th to 75th(box) and 10th to 90th (whiskers) percentiles for immunohistochemicalgrading (0-3) of microglia (E, F, G). One day after surgery mice showedsignificantly higher levels of reactive microgliosis compared to naive,anaesthetics only or surgical mice treated with minocycline (E). Threedays after surgery mice continued to show an increase in reactivemicroglia compared with naive animals (F). By 7 days microglialactivation had returned to normal (G). **p<0.01 and *p<0.05 vs naïveanimals; #p<0.05 vs anesthesia group; †p<0.05 vs minocycline group; forsignificant group difference, (n=7).

FIG. 4. Hippocampal-dependent recall of fear memories is impaired aftersurgery. Rodents underwent fear conditioning and 30 min later they weredivided to receive anesthetics (Anesthesia), or surgery of the tibiaunder anesthesia (Surgery), or the same surgical procedure withminocycline (Minocycline) or enrofloxacin (Enrofloxacin) administration,respectively. Naive group received no treatment. Contextual andacoustic-cued memories were tested three days later. A. Recall ofcontextual delay fear conditioning memories, as measured by freezingbehavior, was impaired in surgical animals compared to naïve andanesthesia groups. Administration of minocycline, but not enrofloxacin,mitigated the surgery-induced, decrement in freezing. *p<0.05 vs naïve,anesthesia and minocycline groups; (n=34). B. Freezing in theauditory-cued test after delay fear conditioning. There was nodifference between the groups in either baseline orauditory-cued-related freezing behavior, thus suggesting thatamygdalar-dependent memory function is intact after surgery. (n=34). C.Freezing to context after trace fear conditioning. Mice subjected tosurgery exhibited reduced freezing to context when compared to naïveanimals, confirming that the inflammation induced by surgery disruptsrecall of fear contextual memories formed in the hippocampus after traceconditioning. *p<0.05; (n=28). D. Hippocampal-dependent, surgery-inducedmemory impairment is shown in the auditory-cued test, in mice trainedwith trace fear conditioning. There is a significant difference betweenthe groups in auditory-cued-related freezing behavior, suggestingdisruption of auditory-cued, hippocampal-dependent, retrieval ofmemories after surgery. No difference was shown in the baseline freezingbehavior. *p<0.05; (n=28).

FIG. 5. Surgery-induced inflammation is mitigated in mice in which IL-1signalling is disabled or reduced.

Immunohistochemistry of hippocampal microglia with anti-CD11b inIL1R^(−/−) mice (A-B). Representative microglia from naïve (A) andsurgical IL-1R^(−/−) mice 24 h after surgery. Scale bar 30 μm. Box andwhiskers plot of microgliosis in IL1R^(−/−) mice (C). Grading ofmicrogliosis confirms that surgery did not activate microglia inIL1R^(−/−) mice, compared to naïve littermates, one day after theprocedure. Circulating IL-1β in IL1R^(−/−) mice and in WT pre-treatedwith IL-1R antagonist prior to surgery (D). Surgery did not induce asignificant increase of IL-1β at 24 h in either IL1R^(−/−) mice or in WTanimals treated with IL-1Ra prior to surgery. Data are expressed as mean±SEM, ***p<0.001 vs any other group; n=6. N WT=naïve wild type; SWT=wild type undergoing surgery; S WT RA=wild type pre-treated withIL-1Ra prior to surgery; S IL1R−/−=mice lacking IL-1R undergoingsurgery; N IL1R−/−=naïve mice lacking IL-1R.

FIG. 6. IL-1 receptor antagonist (IL-1Ra) prevents hippocampalneuroinflammation after surgery.

Immunohistochemistry of hippocampal microglia with anti-CD11b in WT micepre-treated with IL-1Ra (A-B). Representative photomicrographs fromnaïve (A) and surgical mice (B) pretreated with IL-1Ra. Scale bar 30 μm.Box and whiskers plot for grading of microgliosis in IL-1Ra treatedsurgical mice (C).Grading of microgliosis confirms that surgery did notactivate microglia if IL-1Ra was given pre-operatively. Hippocampalexpression of IL-1β in IL1Ra pre-treated surgical mice (D).HippocampalIL-1β did not significantly increase in mice treated with IL-1Raundergoing surgery compared to naïve mice. All assessments wereconducted 24 hours after surgery. Data are expressed as mean ±SEM; n=6.

FIG. 7. Surgery-induced impairment of contextual fear memories isprevented by pre-emptive administration of IL-1 receptor antagonist(IL-1Ra). A. IL-1Ra, injected before surgery, significantly reduced thesurgery-induced decrement in freezing behavior. *p<0.05; (n=30). B.Freezing in the auditory-cued test after delay fear conditioning. Therewas no difference between the groups in either baseline orauditory-cued-related freezing behavior, suggesting that neithersurgery, nor IL-1Ra affected amygdalar-dependent memory function (n=30).Data are expressed as mean ±SEM percentage of freezing response.

FIG. 8. Recall of contextual and auditory-cued memories is not affectedif surgery is delayed three days after conditioning. A. Freezing to thecontext in mice undergoing surgery three days after delay fearconditioning. Surgery performed three days after treatment did notaffect freezing to context when compared to naïve animals. (n=28). B.Freezing in the auditory-cued test after training with delay fearconditioning. There was no difference between the groups in eitherbaseline or auditory-cued-related freezing behavior. (n=28).

FIG. 9: Inflammatory response after LPS exposure. Mice were injectedwith LPS at time zero and plasma levels of TNFα, IL-1β, IL-6 and HMGB-1were measured by ELISA. TNFα was increased after 30 minutes and peakedat 2 hours, returning to baseline thereafter (A; *p<0.01; ***p<0.001 vsnaïve). IL-1β was detected after 2 hours from LPS administration andlevels continued to steadily increase until 24 hours (B; ***p<0.001 vsnaïve). IL-6 expression was highly elevated at 2 hours, decreasing at 6hours but still significantly detectable at 24 hours compared to naïveanimals (C; ***p<0.0001; **p<0.001 vs naïve respectively). Levels ofHMGB1 started to increase at day 1 and until day 3 (D; **p<0.001;***p<0.0001 vs naïve). Increased mRNA expression of IL-1β (E) and IL-6(F) was found at 6 hours after peripheral LPS injection in thehippocampus of mice using qPCR (p<0.001 vs naïve); mRNA expressionreturned to normal by day 1. Data are expressed as mean ±SEM (n=6) andcompared by one-way analysis of variance and Student-Newman-Keulsmethod.

FIG. 10: Blocking IL-1 reduces systemic cytokine release. Animalsreceived LPS (LPS) or treatment with IL-1Ra immediately before LPSexposure (RA). Plasma levels of IL-1β and IL-6 were measured by ELISA at2, 6, and 24 hours. Pre-emptive administration of IL-1Ra significantlyreduced the amount of plasma IL-1β at 6 hours (A; *p<0.01 vs LPS) and 24hours (***p<0.001 vs LPS). IL-6 followed a similar trend, with a strongdecrease in plasma concentrates at 6 hours (B; ***p<0.001 vs LPS) and at24 hours (**p<0.001 vs LPS). To corroborate the findings, levels ofIL-1β and IL-6 were measured in IL-1R^(−/−) (−/−) (A-B, ***p<0.0001 and**p<0.001 vs LPS respectively). IL-1Ra or IL-1R^(−/−) had no effects onHMGB-1 release in plasma (C). Data are expressed as mean ±SEM, (n=6) andcompared by one-way and two-way (IL1R^(−/−)) analysis of variance andStudent-Newman-Keuls method.

FIG. 11: Blocking IL-1 reduces microglia activation. Hippocampi wereharvested at days 1, 3, 7 after LPS administration and stained withanti-CD11b. Pictures show CA1 (scale bar 50 μm, 20×) andphotomicrographs were blindly scored and microglia activation was gradedon a scale 0 (lowest)-3 (highest). PANEL 1: LPS. Reactive microglia werefound at days 1 and 3 after LPS injection (B-C) compared to naive (A).Resting microglia (box A, 40×) shifted to a “reactive state” (box B,40×). PANEL 2: IL-1Ra. Reduction in the number of reactive microglia wasobserved after administering IL-1Ra both at days 1 and 3 (E-F), with nochanges from controls (D). PANEL 3: IL-1R^(−/−). Administration of LPSto IL-1R^(−/−) did not induce microglia activation at any time pointassessed (G-H-I).

Median (horizontal bar) with 25th to 75th (box) and 10th to 90th(whiskers) percentiles for immunohistochemical grading (0-3) illustratespanels 1, 2, and 3. One day after LPS administration we found clearmicrogliosis, which was attenuated by IL-1Ra treatment (day 1 **p<0.001vs naïve, day 3 *p<0.05 vs naïve). Significant reduction in microgliosiswas found both after IL-1Ra administration and in IL-1R^(−/−) (n=4). Nonparametric data are presented with Kruskal-Wallis followed by Dunn'stest.

FIG. 12: Contextual fear response is ameliorated by pre-emptive IL-1Ra.Within thirty minutes following training, mice were injected with LPS.Three days later, rodents were exposed to the same context in which fearconditioning was previously carried out. Contextual fear responsereveals a clear hippocampal-dependent memory impairment (A, **p<0.005 vsnaive). Pre-treatment with IL-1Ra abolished the main symptoms ofsickness behavior and significantly ameliorated the memory retention atday 3 (A, *p<0.05 vs LPS). The auditory-cued test did not show anydifference between groups or in baseline freezing (B). Data areexpressed as mean ±SEM (n=9 for acute behavior) and compared by one-wayanalysis of variance and Student-Newman-Keuls method.

FIG. 13: Systemic TNFα after surgery. Adult mice underwent surgery ofthe tibia under general anesthesia (Sx). Plasma samples were collectedafter 30 minutes, 1, 2, 6 and 24 hours following intervention andmeasured by ELISA. A positive trend was observed within the 1-hourwindow post surgery. Data are expressed as mean ±SEM n=6 and compared byone-way analysis of variance and Student-Newman-Keuls method. S=surgery

FIG. 14: Effects of anti-TNFα prophylaxis. Adult mice underwent surgeryof the tibia under general anesthesia (Sx), or the same surgicalprocedure with anti-TNFα prophylaxis 18 hours prior to surgery (Ab). Thecontrol group was composed of naïve animals. Preemptive administrationof anti-TNFα reduced the amount of systemic IL-1β as measure by ELISAboth at 6 hours and 24 hours post intervention (A, **p<0.01, ***p<0.001compared to sx respectively). Anti-TNFα prophylaxis also reduced thesystemic levels of IL-6 both at 6 and 24 hours following surgery (B,**p<0.01, *p<0.05 compared to sx respectively). Delayed administrationof anti-TNFα (legend D) resulted in no changes from surgery assessingboth IL-1β and IL-6. Tibia surgery resulted in increased hippocampallevels of IL-1β, which was successfully reduced following treatment (C,**p<0.01 vs sx). Immunohistochemistry of microglia in the hippocampuswith anti-CD11b one day after surgery. Pictures show CA1 (scale bar 50μm, 20×) and photomicrographs were blindly scored and microgliaactivation was graded on a scale 0 (lowest)-3 (highest). Neither naïve(D) or mice treated with anti-TNFα (F) showed evidence of reactivemicrogliosis. Reactive microglia were found in animals undergoingsurgery (E). Median (horizontal bar) with 25th to 75th (box) and 10th to90th (whiskers) percentiles for immunohistochemical grading (0-3) ispresented for data illustration (G). One day after surgery there is anevident reduction in microgliosis following therapy (**p<0.01 vs sx).Contextual fear response reveals clear hippocampal-dependent memoryimpairment (H, *p<0.05 vs naive). Pre-treatment with anti-TNFαameliorates the memory retention (*p<0.05 vs sx). Data are expressed asmean ±SEM n=6 (n=10 for acute behavior) and compared by one-way analysisof variance and Student-Newman-Keuls method. Non parametric data arepresented with Kruskal-Wallis followed by Dunn's test. Sx=surgery,Ab=antibody, D=delayed administration of antibody

FIG. 15: Roles for both IL-1β and TNFα. Adult MyD88^(−/−) mice underwentsurgery of the tibia under general anesthesia (Sx). The control groupwas composed of MyD88^(−/−) naïve animals. Systemic levels of IL-1β (A,++p<0.001, +p<0.01 vs surgery WT respectively) and IL-6 (B, +++p<0.001,++p<0.001 vs surgery WT respectively) were significantly reducedfollowing surgery in MyD88^(−/−) both at 6 and 24 hours. No changes inhippocampal levels of IL-1β were reported (C). Neither naïve (D) norMyD88^(−/−) undergoing surgery (E) showed evidence of reactivemicrogliosis. Median (horizontal bar) with 25th to 75th (box) and 10thto 90th (whiskers) percentiles for immunohistochemical grading (0-3) ispresented for data illustration (F). Contextual fear response reveals nohippocampal-dependent memory impairment following surgery in MyD88^(−/−)(G). Adult MyD88^(−/−) mice underwent surgery of the tibia under generalanesthesia with anti-TNFα prophylaxis 18 hours prior to surgery (Ab).Preemptive administration of anti-TNFα reduced the amount of systemicIL-1β as measure by ELISA to baseline both at 6 hours and 24 hours postintervention (H, ***p<0.001, *p<0.01, compared to sx respectively).Levels of IL-6 were also measured, there was a similar reduction withvalues back to baseline at both time points (H, ***p<0.001, **p<0.01,compared to sx respectively). Data are expressed as mean ±SEM n=6 (n=10for acute behavior) and compared by one-way analysis of variance andStudent-Newman-Keuls method. Non parametric data are presented withKruskal-Wallis followed by Dunn's test. Sx=surgery, Ab=antibody,D=delayed administration of antibody

FIG. 16: Anti-TNFα prophylaxis in TLR4^(−/−). Adult TLR4^(−/−) miceunderwent surgery of the tibia under general anesthesia (Sx), or thesame surgical procedure with anti-TNFα prophylaxis 18 hours prior tosurgery (Ab). The control group was composed of TLR4^(−/−) naïveanimals. Preemptive administration of anti-TNFα reduced the amount ofsystemic IL-1β as measure by ELISA to baseline both at 6 hours and 24hours post intervention (A, **p<0.01, ***p<0.001, compared to sxrespectively). There was a similar reduction in levels of IL-6, withvalues back to baseline at both time points (B, ***p<0.001, **p<0.01,compared to sx respectively). TLR4^(−/−) showed signs ofneuroinflammation but levels of hippocampal IL-1β were reduced byanti-TNFα prophylaxis (C, *p<0.01 vs sx). Immunohistochemistry ofmicroglia in the hippocampus with anti-CD11b one day after surgery.Pictures show CA1 (scale bar 50 μm, 20×) and photomicrographs wereblindly scored and microglia activation was graded on a scale 0(lowest)-3 (highest). Neither naïve (D) or mice treated with anti-TNFα(F) showed evidence of reactive microgliosis. Reactive microglia werefound in TLR4^(−/−) undergoing surgery (E, **p<0.01 vs naïve and abgroups). Median (horizontal bar) with 25th to 75th (box) and 10th to90th (whiskers) percentiles for immunohistochemical grading (0-3) ispresented for data illustration (G). Contextual fear response revealsclear hippocampal-dependent memory impairment similar to WT (H, *p<0.05vs naive). Data are expressed as mean ±SEM n=6 (n=10 for acute behavior)and compared by one-way analysis of variance and Student-Newman-Keulsmethod. Non parametric data are presented with Kruskal-Wallis followedby Dunn's test. Sx=surgery, Ab=antibody

FIG. 17: Effects of postsurgical LPS on neuroinflammation and behavior.Hippocampi were harvested at days 3 and 7 after surgery and stained withanti-CD11b. Pictures show CA1 (scale bar 50 μm, 20×) andphotomicrographs were blindly scored and microglia activation was gradedon a scale 0 (lowest)-3 (highest). PANEL 1: SURGERY. Reactive microgliawere found at postoperative day (POD) 3, returning to normal by day 7(B-C) compared to naïve (A). PANEL 2: SURGERY+LPS. Moderate and mildmicrogliosis was observed at days 3 and 7, respectively (E-F), comparedto control (D). PANEL 3: LPS. Reactive microglia were found at day 3after LPS injection (H) with no significant changes at day 7 (I),compared to untreated animals (G). Immunohistochemical grading (0-3)illustrates panels 1 and 2. At POD 3 there was a significant differencebetween surgery and surgery+LPS groups (p<0.05). At POD 7 mildmicrogliosis was reported following LPS administration (*p<0.05 vscontrol) (n=4). Non parametric data are presented with Kruskal-Wallisfollowed by Dunn's test. Contextual fear response, as measured byfreezing behavior, is also impaired in animals receiving surgeryfollowed by LPS exposure compared to naïve and surgery groups (G)(**p<0.05 vs surgery). Data are expressed as mean ±SEM (n=10) andcompared by one-way analysis of variance and Student-Newman-Keuls methodand student t-test for comparison between surgery and surgery with LPS.Mice were injected with LPS (1 mg/kg) 24 h following surgery. Levels ofplasma IL-1β were measured by ELISA. At 72 h following surgery, LPStreated animals had a sustained elevation in IL-1β (H; **p<0.01 vscontrol). No IL-1β was detected after surgery or LPS only at 72 h. Dataare expressed as mean ±SEM (n=4) and compared by one-way analysis ofvariance and Student-Newman-Keuls method with Bonferroni corrections.

EXAMPLE 1 Systemic and Hippocampal IL-1β-Mediated Inflammation UnderlieCognitive Dysfunction Following Surgery

While post-operative cognitive dysfunction (POCD) often complicatesrecovery from major surgery, the pathogenic mechanisms remain unknown.We explored whether systemic inflammation, in response to surgicaltrauma, triggers hippocampal inflammation and subsequent memoryimpairment, in a mouse model of orthopedic surgery. Wild type and KOmice (lacking IL-1β receptor, IL-1R^(−/−)) underwent surgery of thetibia under general anesthesia. Separate cohorts of animals were testedfor memory function with fear conditioning tests, or euthanized atdifferent times to assess levels of systemic and hippocampal cytokinesand microglial activation; the effects of interventions, designed tointerrupt inflammation (specifically and non-specifically), were alsoassessed. Surgery caused hippocampal-dependent, memory impairment thatwas associated with increased plasma cytokines, as well as reactivemicrogliosis and IL-1β transcription and expression in the hippocampus.Non-specific attenuation of innate immunity with minocycline preventedsurgery-induced changes. Functional inhibition of IL-1β, both inIL-1R^(−/−), and in wild type mice pretreated with IL-1 receptorantagonist (IL-1Ra), mitigated the neuroinflammatory effects of surgeryand memory dysfunction.

Our results suggest that a peripheral surgery-induced innate immuneresponse triggers an IL-1β-mediated inflammatory process in thehippocampus that underlies memory impairment. This may represent aviable target to interrupt the pathogenesis of post-operative cognitivedysfunction.

Abbreviations

CNS=central nervous system; CS=conditional stimulus; ELISA=enzyme linkedimmunosorbent assay; IFN=interferon; IL=interleukin; IL-1R^(−/−)=Notexpressing IL-1 receptor; IL-1Ra=interleukin-1 receptor antagonist;ir=immunoreactive; i.p.=intraperitoneal; i.v.=intravenous; KO=knock out;LPS=lipopolysaccharide; MAC=minimum alveolar concentration; MAPK=mitogenactivated protein kinase; MHC=major histocompatibility complex;PKC=protein kinase C; POCD=post-operative cognitive dysfunction;qRT-PCR=quantitative real time polymerase chain reaction;s.c.=subcutaneous; TNF=tumor necrosis factor

Material and Methods

All the experiments were conducted under Home Office approved licenceand were performed using 12-14 weeks old male C57-BL6 mice (Harlan,Oxon, UK). IL-1R^(−/−) mice, kindly provided by Professor NancyRothwell,¹⁷ were bred in house on a C57BL/6 background and age-matchedto wild type counterparts.(For further details please refer tosupplemental methods).

Surgery and Pharmacological Treatments

Mice were subjected to an open tibial fracture of the left hind paw withintramedullary fixation in aseptic conditions under general anaesthesiawith isoflurane and analgesia with buprenorphineas previouslydescribed¹⁸. Other groups of animals were not subjected to anyintervention (naïve), or received anesthetic/analgesia alone, orunderwent surgery with concurrent administration of minocycline,enrofloxacin, or IL-1 receptor antagonist (IL-1Ra). (For further detailsplease refer to supplemental methods).

Real Time PCR (qRT-PCR)

Total RNA was extracted using RNeasy Kit (Qiagen) and quantified. Theone-step qRT-PCR was performed on a Rotor-Gene 6000 (Corbett LifeScience), using Assay-On-Demand premixed Taqmanprobe master mixes(Applied Biosystems). Results are expressed as fold-change. (For furtherdetails please refer to supplemental methods).

Cytokine Measurement

IL-6, TNF-α and IL-1β were measured by ELISA¹⁹ (Biosource, CA; BenderMedsystem, CA, respectively). Hippocampal IL-1β was measured by ELISA(Bender Medsystem, CA), as previously described²⁰. To confirmreliability of dilution linearity and spike recovery cytokinemeasurement were also performed in mice in which inflammation wasinduced with i.p. LPS (0111:B4, Invivogen, CA).(For further detailsplease refer to supplemental methods).

Immunohistochemistry

Fixed brains were collected for immunohistochemical DAB staining forCD11b and scored as previously described²¹. (For further details pleaserefer to supplemental methods).

Fear Conditioning Tests

Mice were conditioned 30 minutes or 3 days prior to intervention bytraining with two cycles of tone and foot-shock pairings. In delay fearconditioning a foot-shock was administered during the last 2 seconds ofeach 20 seconds-lasting tone. In trace fear conditioning, the foot-shockonset, lasting 2 seconds as in the previous paradigm, followed a20-second gap after each tone termination. Three days afterconditioning, mice were placed back in the original conditioning chamberfor 270 seconds, where no tone or shock were presented, to assess recallof contextual fear memory. After 3 h mice were placed in a novelenvironment (different context from training) to test for auditory-cuedmemory. Following an initial baseline period of 135 seconds during whichfreezing was scored in absence of noise, the auditory cue was presentedfor the final 135 seconds of the test, and freezing was again recorded.(For further details please refer to supplemental methods).

Data Analysis

Data are expressed as mean ±SEM. Statistical analysis was performed withanalysis of variance followed by the Student-Newman-Keuls test fornumerical data. Student's t test was only used for comparisons betweentwo groups. The non-parametric test of Kruskal-Wallis followed by theDunn's test was used for categorical data. A p value <0.05 wasconsidered to be of statistical significance.

Results Surgery Elevates Plasma Concentration of Inflammatory CytokinesIL-1β and IL-6

Plasma IL-1β and IL-6 were unchanged at 2 hours; these peaked at 6 hours(FIG. 1, A-B) increasing by 7-(IL-1β: 42.63 pg/ml, SEM ±9.60, n=6,p<0.001) and 20-fold (IL-6: 128.50 pg/ml, SEM ±19.64, n=6, p<0.001)above baseline levels, respectively. At 24 h post-surgery, IL-1β andIL-6 were increased 6-(IL-1β: 36.90 pg/ml, SEM ±6.54, n=6, p<0.001) and5-fold (IL-6: 31.74 pg/ml, SEM ±5.28, n=6, p<0.05), respectively,compared with naïve animals (IL-1β: 6.09 pg/ml, SEM ±1.31, n=6; IL-6:5.89 pg/ml, SEM ±2.10, n=6) (FIG. 1, A-B). TNF-α remained undetectableat all time points under all conditions (data not shown). Theadministration of anesthetics alone produced no change of cytokines fromthe baseline levels observed in naïve mice (FIG. 1, A-B). Pre-operativeadministration of minocycline, an antimicrobial with anti-inflammatoryproperties²², reduced the plasma concentrations of cytokines back topre-surgery levels (FIG. 1, A-B). Conversely enrofloxacin, anantimicrobial with a similar broad-spectrum to that of minocycline butdevoid ofanti-inflammatory activity, exerted no effect on IL-1β plasmaconcentration in mice undergoing surgery (FIG. 1, A).

Surgery Increases Hippocampal Cytokines

Hippocampal IL-1β and IL-6 transcription increased 2-fold and 4-foldrespectively following surgery at 6 h (FIG. 2, A-B). Consistently, whenthe expression of hippocampal IL-1β was assessed 6 hours after surgery,there was a 2-fold increase of IL-1β levels (5.53 pg/100 μg of proteins,SEM ±0.89, n=7, p<0.05) compared to naïve counterparts (2.73 pg/100 μgof proteins, SEM ±0.39, n=7) (FIG. 1, C). IL-1β expression in thehippocampus was not changed in animals exposed to anesthesia alone, frombaseline. Minocycline, but not enrofloxacin, reduced IL-1β expression insurgical animals to naïve levels.

Surgery Activates Microglia

LPS-injected mice (positive inflammatory control) exhibited CD11bimmunoreactive (ir) microglia in an activated morphologic phenotypecharacterized by hypertrophy of cell bodies, retraction of processes, anapparent amoeboid morphology, and increased levels of immunoreactivity.Surgery induced similar morphological changes of microglial reactivityat 24 h (FIG. 3) that was significantly different from that of naïve(n=7, p<0.01) and anesthesia-only-treated animals (n=7, p<0.05).Surgery-induced reactive microgliosis was reduced by day 3 (FIG. 3, F)and returned to baseline by 7 days (FIG. 3, G). Administration ofminocycline to surgical mice prevented reactive microgliosis (FIG. 3,E-F).

Surgery Impairs Contextual Fear after Delay Conditioning

As expected, there was no difference in freezing time between the groupsduring training (data not shown). Surgery performed 30 min followingtraining significantly reduced freezing to context that was mitigated byminocycline, but not by enrofloxacin (FIG. 4, A); anesthesia aloneproduced no change. All the animals displayed the same freezing behaviorwhen exposed to the tone (FIG. 4, B).

Surgery Impairs Memory for Tone in Trace Fear Conditioning

As opposed to delay fear conditioning, trace fear conditioning imposes abrief gap between the tone termination and shock onset. Trace and delayfear conditioning differ in that, in trace, the fear response to boththe tone and context highly depends on hippocampal integrity²³. Whenexposed to the context, mice undergoing surgery demonstrated asignificant reduction of freezing behavior compared with naïvelittermates (n=28, p<0.05), consistent with the results from the delayparadigm (FIG. 4, C). Moreover, when the auditory-cued test was carriedout, there was a significant difference between surgical and naïvesubjects in auditory cued-dependent freezing (n=28, p<0.05), with areduction in surgical animals (FIG. 4, D).

Surgery does not Induce Inflammation in IL-1R^(−/−) Mice or Mice Treatedwith IL-1Ra

Surgery did not increase circulating IL-1β or hippocampal microgliosisin mice lacking the IL-1 receptor (FIG. 5, A-B-C-D) or in micepretreated with IL-1Ra. Similarly, surgery did not increase hippocampalmicrogliosis (FIG. 6, A-B-C) or expression of IL-1β in IL-1Ra treatedsurgical mice (FIG. 6, D). In the delay fear conditioning paradigm,pre-treatment with IL-1Ra prevented the decrement in postoperativefreezing behavior (FIG. 7, A). Results from the auditory-cued test onthese groups showed no difference, thereby confirming previous evidencefrom this study that the amygdala is not functionally impaired bysurgery or by IL-1Ra administration (FIG. 7, B).

Contextual Fear Memory is not Impaired in Surgical Animals UndergoingSurgery Three Days after Delay Conditioning

The data thus far indicate that hippocampal-dependent retrograde amnesiadevelops when surgery takes place 30 minutes after training. In order todetermine if the memory impairment was caused by interruptedconsolidation as opposed to a more permanent loss of hippocampalfunction, mice underwent surgery 72 hours after delay fear conditioning.The animals were tested for both tone and context memory 3 days later,following the same surgery-to-context time delay as in the previoustests. The test for contextual and acoustic-cued memory showed nostatistical difference between surgical and naïve animals (n=28) (FIG.8, A-B).

Discussion

Data from these studies suggest that inflammation plays a pivotal rolein the pathogenesis of POCD as evidenced by the protection affordedsurgical animals by minocycline, a non-specific inhibitor ofinflammation. Also, we demonstrated that IL-1β is likely to have acausal role in conveying the inflammatory signal from a peripheralsurgical site to the brain. Hippocampal inflammation follows peripheralsurgery as demonstrated by a local increase in the transcription andexpression of IL-1β as well as reactive microgliosis. We show thatattenuation of the IL-1β response to surgery prevents post-operativememory dysfunction. Post-surgical impairments for contextual fear memoryand for auditory-cued memory in trace, but not for auditory-cued memoryin delay conditioning, also suggest that postoperative memorydysfunction is derived from the hippocampus rather than other componentsof the fear circuit, such as auditory thalamic, amygdalar orperiaqueductal gray regions.

The impaired hippocampal-dependent contextual fear memory after asepticsurgery is similar to the impairment in contextual fear conditioningthat follows intraperitoneal administration of LPS in a model ofperipheral inflammation caused by infection²⁴. Also, neither LPS²⁴ norsurgery disrupts delay auditory-cued memories. That the hippocampus iscritical for post-surgery cognitive impairment is confirmed by the traceconditioning procedure in which impaired freezing responses wereobserved to both context and tone.

A possible causality relationship between surgery, inflammation andmemory impairment was suggested by the effects of minocyclineadministration in reducing surgery-induced peripheral and hippocampalcytokine expression, reactive microgliosis and behavioral impairment;minocycline also restored behavioral impairment in a mouse model ofLPS-induced inflammation²⁵. Minocycline reduces microglial activationthrough the inhibition of interferon (IFN)-γ-induced PKCα/βIIphosphorylation and both PKCα/βII and IRF-1 nuclear translocation,ultimately converging in the partial down regulation of MHC IIproteins²⁶. Importantly, minocycline inhibits the transcription factorp38-MAPK, which plays a pivotal role in the cascade leading to thebiosynthesis of cytokines such as IL-1β and IL-6 as well as otherpro-inflammatory mediators²⁷. Moreover, at a functional level,minocycline appears to improve behavioral performance in a mouse modelof spatial learning and memory through reduction of microglialactivation²⁸.

Despite the aseptic technique employed, it is possible to ascribe theadvent of post-operative hippocampal inflammation and cognitivedysfunction and its attenuation by an antimicrobial (minocycline) to aninfective process following surgical intervention. However, in thisstudy no clinical evidence of infection was seen in any of the animalsas shown in previous studies¹⁸. Importantly, in our study, theadministration of enrofloxacin, a wide spectrum antibiotic often used inrodents but devoid of any anti-inflammatory properties, did not improveany of the surgery-induced effects.

It could be argued that pain may be a confounding factor producingimmobility and thus influencing the extent of “freezing” in thebehavioral tests. However, our model of surgery aimed to reproduceroutine clinical settings and, accordingly, administration of ananalgesic opiate (buprenorphine) in our experiments likely mitigatedsurgical pain. If nociceptive input caused the animals to restrictmovement of their affected limb during retrieval of context orauditory-cued memories, we would have expected to see more, and notless, freezing. It is of some importance to note that even pain producedby subcutaneous injection of formalin into a hind paw does not disruptfreezing seen with contextual fear conditioning²⁹. Moreover, a possibleinterference from pain in the extinction tests was addressed by thetesting for memory impairment in the setting in which surgery wasdelayed by three days after conditioning. Since the delay betweensurgery and retrieval tests in this experiment was the same as in theother implemented fear conditioning experiments, it is reasonable toconclude, based on results showing no differences between surgical andnaïve animals in memory retrieval, that there was no interference frompost-operative pain in all the fear conditioning retrieval testsemployed in this study.

In the activated state, microglia are capable of mountingmacrophage-type innate immunity and secrete pro-inflammatory cytokines,reactive oxygen species, excitotoxins (such as glutamate) andneurotoxins such as β-amyloid precursor protein³⁰. Activation ofmicroglia has been linked to the cognitive dysfunction that is seen insickness behavior and is causally related to impairment of long-termpotentiation in advanced age³¹. Thus, increased microglial reactivity,and the associated inflammatory processes, are capable of producing themolecular changes that attenuate signalling pathways involved in memoryformation³².

We have demonstrated that IL-1β plays a pivotal role in surgery-inducedcognitive dysfunction^(24,33). Peripheral cytokines can signal to thebrain via both blood and neural routes thereby stimulating cytokineproduction by glial cells^(34,35,36), especially in thehippocampus^(37,20). Evidence of increased hippocampal transcription ofIL-1β suggests the possible role of microglia in the de novo productionof cytokines in our mouse model. The specificity of IL-1β involvement isemphasized by the experiments involving IL-1R^(−/−) mice and wild typemice treated with IL-1Ra in which microglial activation in thehippocampus is no longer triggered after surgery. IL-1β interferes withhippocampal long-term potentiation^(19,38) that has been viewed as anessential electrophysiological correlate of memory³⁹. IL-1β acts eitherdirectly, or indirectly through microglial activation, on theintracellular neuronal mechanisms that stabilize the long-termplasticity necessary for memory such as protein synthesis. Loss ofmemory induced by IL-1β is unlikely to be caused by permanent damage,retrieval failure or an inability to perform the freezing response, assuch deficits would have also appeared when surgery occurred thee daysafter training.

We have shown that elevated systemic and brain tissue cytokines, as wellas hippocampal microglial activation, are all reduced by treatment withperipherally injected minocycline or IL-1Ra. While these data suggestthat humoral, rather than, or in addition to, neural factors areinvolved⁴², further studies are required. Attenuation of theneuroinflammatory process with either minocycline or by interferencewith IL-1β signalling prevents the post-surgical cognitive dysfunction.This neuroinflammatory process and its initiation, represents arealistic target for therapeutic interventions with major potentialbenefits for the ageing surgical population. Subsequent studies toelucidate whether the neuroinflammatory response can be modulated byanesthetic agents or by selective anti-inflammatory strategies may behelpful in ameliorating the adverse consequences of post-operativecognitive decline.

Supplementary Material Animals and Surgical Methods

All the experiments were conducted under Home Office approved licenceand were performed using 12-14 weeks old male C57-BL6 mice (Harlan,Oxon, UK) housed in groups of up to 4 animals/cage, under a 14:10 hourslight-dark cycle, in a constant temperature and humidity controlledenvironment, with free access to food and water. Acclimation tolight/dark cycle for a minimum of 7 days preceded any intervention orassessment. All the animals were checked on a daily basis for signs ofinfection. Evidence of poor grooming, huddling, piloerection, weightloss, wound dehiscence, muscle twitching, back arching and abnormalactivity, were recorded.

IL-1R^(−/−), kindly provided by Professor Nancy Rothwell, University ofManchester, were generated as previously described¹, were bred in houseon a C57BL/6 background and age-matched to wild type counterparts.

Mice were randomly assigned to the following groups: surgery undergeneral anesthesia (S), surgery under general anesthesia plusminocycline (M), surgery under general anesthesia plus enrofloxacin (E),surgery under general anesthesia plus interleukin-1 receptor antagonist(IL-1Ra) (I), surgery in IL1R^(−/−) mice under general anesthesia (K),general anesthesia without surgery (A), and no intervention (naïvewild-type, or naïve IL1R^(−/−)). Under aseptic conditions, groups ofmice were subjected to an open tibial fracture of the left hind paw withan intramedullary fixation as previously described². Briefly, isoflurane(Abbot Laboratories Ltd., Queensborough, Kent, UK) 2.1% in air (1.5MAC)³ and buprenorphine (Reckitt Benckiser Healthcare Ltd, Hull, UK) 0.1mg/kg subcutaneously (sc), were given to provide both surgicalanesthesia and extended post-operative analgesia for the surgicalintervention and post-surgical recovery. After shaving the overlyingskin and disinfecting with chlorhexidine gluconate 0.5% and isopropylalcohol 70% (PDI, Orangeburg, N.Y., USA) a fracture of the tibial shaftwas created under direct vision. A longitudinal incision was madethrough the skin and fascia lateral to the tibia to expose the bone. A0.5 mm hole was drilled just above the proximal third of the tibia toinsert an intramedullary 0.38 mm diameter stainless steel fixation wire.Subsequently, the fibula and the muscles surrounding the tibia wereisolated, the periosteum stripped over a distance of 10 mmcircumferentially and an osteotomy was performed with scissors at thejunction of the middle and distal third of the tibia. The skin wassutured with 8/0 Prolene and intra-operative fluid loss was replacedwith 0.5 ml of subcutaneously injected normal saline. Mice in groups M,E and I also received an intraperitoneal (i.p.) injection of minocycline(Sigma, Poole, UK) 40 mg/kg 2 hours prior to surgery and once dailyuntil assessment of outcome, or enrofloxacin 10 mg/kg 2 hours prior tosurgery and twice daily thereafter until assessment of outcome or IL-1Ra100 mg/kg prior to surgery, respectively. Mice in group A receivedanesthesia (isoflurane 1.5 MAC for 20 minutes) and analgesia(buprenorphine 0.1 mg/kg) with no surgical intervention or othertreatment. All the animals were injected twice daily with experimentaldrugs or equivalent volumes (0.1 ml) of saline. Mice were allowed free,unrestricted food and water intake following recovery from theprocedure. Mice from each treatment group were randomly assigned to twodifferent assessment groups for either harvesting blood and tissuesamples or for cognitive behavior, in order to obviate possibleconfounding effects of fear conditioning testing⁴ on inflammatorymarkers.

Conditioning Chamber and Fear Conditioning

Fear conditioning is used to assess learning and memory in rodents,which are trained to associate a conditional stimulus (CS), such as atone, with an aversive, unconditional stimulus (US), such as afoot-shock⁵. Freezing behavior is an indicator of aversive memory thatis measured when subjects are re-exposed to the CS. The environment, orcontext, in which the animals are trained, represents a moresophisticated example of CS. In delay fear conditioning, where the toneand shock are temporally contiguous, lesions of the amygdala disruptrecall of fear responses to both auditory cue and context, whereaslesions of the hippocampus disrupt context-related but not auditorycue-related memories^(5,6).

The behavioral study was conducted using a conditioning chamber (Med.Associates Inc., St. Albans, Vt., USA). The back- and the side-walls ofthe chamber were made of aluminium, whereas the front door and theceiling were of transparent Plexiglas. The floor of the chamberconsisted of 36 stainless steel rods (1 mm diameter) spaced 0.5 cm apart(center-to-center). The rods were wired to a shock generator andscrambler for the delivery of foot shock. Prior to testing, the chamberswere cleaned with a 5% sodium hydroxide solution. Background noise (60dB, A scale) was provided by means of a fan positioned on one of thesidewalls. An infrared video camera, mounted in front of the chambercaptured behavior (Video Freeze, Med. Associates Inc., St. Albans, Vt.,USA). In order to make the environment (context) different foracoustic-cued behavioural assessment, the shape of the chamber waschanged from rectangular to triangular, the ceiling colour was blackinstead of white, the flooring rods were covered with a smooth surfaceand background noise was removed. Assessment of the acoustic-cuedbehavior of tone-to-shock pairings in the novel context tests amygdalar,rather than hippocampal function⁷. Video data were collected by aninfrared camera positioned in front of the chamber and was stored in acomputer compatible format (Video Freeze, Med. Associates Inc., St.Albans, Vt., USA). On each day of testing, mice were transported to thebehavioral room and left undisturbed for at least 20 minutes beforeplacing them into the conditioning chamber. Freezing was recognized bythe software as a total lack of movement excluding breathing butincluding movement of fur, vibrissae and skeleton. The percentage oftime spent freezing over the total time spent in the chamber toaccomplish the test was used to score memory and learning abilities. Adecrease in the percentage of time spent freezing indicated impairmentof these abilities.

In delay fear conditioning, training consisted of placing the mouse inthe conditioning chamber and allowing exploration of the context for 100seconds. Next, an auditory cue (75-80 dB, 5 kHz), the conditionalstimulus (CS), was presented for 20 seconds. A 2-second foot-shock (0.75mAmp), the unconditional stimulus (US), was administered during thefinal 2 seconds of the CS. This procedure was repeated with aninter-trial interval of 100 seconds, and the mice were removed from thechamber 30 seconds later. Trace fear conditioning differed from delay inthat the 2-second foot-shock was administered 20 seconds aftertermination of the tone. With the termination of the trial, which lasteda total of 270 seconds, every mouse was taken individually to thesurgery room, within 30 minutes. All the animals, regardless to thespecific intervention, underwent the same handling, including naïveanimals. After testing, animals were returned to their housing cage.Three days after conditioning, mice were transported again to thebehavioral room and left undisturbed for 15 minutes. They were returnedinto the same chamber where training had occurred for a context testlasting 270 seconds, during which no tones or foot-shocks weredelivered. Freezing behavior in response to context was recorded by thesoftware. At the end of the test, mice were individually returned totheir home cage. Approximately 3 hours later, freezing was recorded in anovel environment and in response to the auditory cue. The novelenvironment resulted from modifications to the basic chamber consistingof an opaque Plexiglas triangle; a Plexiglas floor; increasedillumination; no background noise from the fan; and a different smell.Mice were placed in this novel environment, and time sampling was usedas baseline during which freezing was scored for 135 seconds. Theauditory cue was then presented for 135 seconds, and freezing was againrecorded.

Quantification of IL-1β Transcripts by Quantitative Real Time PCR(qRT-PCR)

The hippocampus was rapidly extracted under a dissecting microscope,placed in RNA-later solution (Applied Biosystems, Ambion) and stored at4° C. Total RNA was extracted using RNeasy Kit (Qiagen) and quantified.The one-step qRT-PCR was performed on a Rotor-Gene 6000 (Corbett LifeScience), using Assay-On-Demand premixed Taqman probe master mixes(Applied Biosystems). Each RNA sample was run in triplicate, andrelative gene expression was calculated using the comparative thresholdcycle ΔΔCt and normalized to beta-actin (ACTB). Results are expressed asfold-change.

Cytokine Measurement

Blood was collected by cardiac puncture into heparin coated syringeswhilst animals were under terminal anesthesia with pentobarbital.Samples were centrifuged at 3500 rpm for 10 minutes at 4° C. and plasmawas collected and stored frozen at −80° C. until assaying. IL-6 andTNF-α were measured in plasma using a commercially available ELISA kit(Biosource, CA), whereas the ELISA kit for IL-1β was from a differentmanufacturer (Bender Medsystem, CA). The sensitivities of the assayswere 1.2 pg/ml for IL-1β, 3 pg/ml for TNF-α and 3 pg/ml for IL-6.Positive controls consisted of animals treated with i.p. LPS (0111:B4,Invivogen, CA) (data not shown).

Under terminal anesthesia with pentobarbital, each mouse was euthanizedand the brain quickly removed following decapitation. The hippocampuswas dissected under microscopy on a frosted glass plate placed on top ofcrushed ice, then snap frozen and stored at −80° C. until processing.Each hippocampus was added to Iscove's culture medium containing 5%fetal calf serum (FCS) and a cocktail of enzyme inhibitors (in mM: 100amino-n-caproic acid, 10 EDTA, 5 benzamidine-HCl, and 0.2phenylmethylsulfonyl fluoride). The proteins were mechanicallydissociated from tissue by means of sonication in a container plunged inice. This consisted of 3 cycles of cell disruption each lasting 3seconds. Sonicated samples were centrifuged at 10000 rpm for 10 minutesat 4° C. Supernatants were collected and stored at −80° C. until theELISA was carried out. IL-1β was measured in the supernatant fromhippocampal extracts, which were appropriately diluted prior tomeasurement to fall on the linear portion of the sigmoid curve, using acommercially available ELISA kit (Bender Medsystem, CA). The sensitivityof the assay was 1.2 pg/ml. The ELISA kit was validated for use withbrain tissues. Samples collected from mice treated with LPS (0111:B4,Invivogen, CA) 3 mg/kg i.p. were used to confirm reliability of dilutionlinearity and spike recovery (data not shown).

Immunohistochemistry

Under terminal anesthesia with pentobarbital, mice were euthanized andperfused transcardially with ice-cold heparinized 0.1M phosphate buffersolution (PBS) followed by 4% paraformaldehyde in 0.1M PBS at pH 7.4(VWR International, Lutterworth, Leicester, UK). The brains wereharvested and post-fixed in 4% paraformaldehyde in 0.1M PBS at 4° C. andcryoprotected in 0.1M PBS solutions containing 15% sucrose for 24 hours(VWR International, Lutterworth, Leicester, UK) and then 30% sucrose fora further 48 hours.

Brain tissue was freeze-mounted in OCT embedding medium (VWRInternational, Lutterworth, Leicester, UK). 30 μm thick coronal sectionsof the hippocampus were cut sequentially in groups of 6 and mounted onSuperfrost® plus slides (Menzel-Glaser, Braunschweig, Germany).

The rat anti-mouse monoclonal antibody, anti-CD11b (low endotoxin, cloneM1/70.15) in the concentration of 1:200 (Serotec, Oxford, UK) was usedto label microglia. Visualization of immunoreactivity for CD11b wasachieved using the avidin-biotin technique (Vector Labs, Cambridge, UK)and a goat anti-rat secondary antibody (Chemicon International, CA, USA)at a concentration of 1:200. A negative control omitting the primaryantibody was performed in all experiments. A positive control groupconsisted of animals injected i.p. with LPS 3 mg/kg⁸.Immunohistochemical photomicrographs were obtained with an Olympus BX-60microscope and captured with a Zeiss KS-300 colour 3CCD camera. Theassessment of staining, by an observer that was blinded to theinterventional group, was based upon a 4-point categorical scalemodified from Colburn and colleagues⁹, which uses a combined evaluationof the level of microglial activation from both cell morphology andimmunoreactivity.

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REFERENCES FOR SUPPLEMENTAL METHODS

-   1. Labow M, Shuster D, Zetterstrom M, Nunes P, Terry R, Cullinan E    B, et al. Absence of IL-1 signaling and reduced inflammatory    response in IL-1 type I receptor-deficient mice. J Immunol 1997;    159: 2452-61.-   2. Harry L E, Sandison A, Paleolog E M, Hansen U, Pearse M F,    Nanchahal J. Comparison of the healing of open tibial fractures    covered with either muscle or fasciocutaneous tissue in a murine    model. J Orth Res 2008; 26: 1238-44.-   3. Engelhardt T, Lowe P R, Galley H F, Webster N R Inhibition of    neuronal nitric oxide synthase reduces isoflurane MAC and motor    activity even in nNOS knockout mice. Br J of Anesth 2006; 3: 361-6.-   4. Nguyen K T, Deak T, Owens S M, Kohno T, Fleshner M, Watkins L R,    Maier S. F. Exposure to acute stress induces brain IL-1b protein in    the rat. J Neurosci 1998; 18: 2239-46.-   5. Kim J J & Fanselow M S. Modality-specific retrograde amnesia of    fear. Science. 1992; 256: 675-7.-   6. Phillips R G & LeDoux J E Differential contribution of amygdala    and hippocampus to cued and contextual fear conditioning. Behav    Neurosci. 1992; 106: 274-85.-   7. Quinn J J, Oommen S S, Morrison G E, Fanselow M S. Post-training    excitotoxic lesions of the dorsal hippocampus attenuate forward    trace, backward trace, and delay fear conditioning in a temporally    specific manner. Hippocampus 2002; 12: 495-504.-   8. Qin L, Wu X, Block M L, Liu Y, Breese G R, Hong J S, et al.    Systemic LPS causes chronic neuroinflammation and progressive    neurodegeneration. Glia 2007; 55: 453-62.-   9. Colburn R W, DeLeo J A, Rickman A J, Yeager M P, Kwon P, Hickey    W F. Dissociation of microglial activation and neuropathic pain    behaviors following peripheral nerve injury in the rat. J    Neuroimmunol 1997; 79: 163-75.

EXAMPLE 2 Cytokines Inform Sepsis-Induced Cognitive Dysfunction

The impact of pro-inflammatory cytokines on neuroinflammation andcognitive function after lipopolysaccharide (LPS) challenge remainselusive. Herein we provide evidence that despite a temporal correlationbetween high-mobility group box 1 (HMGB-1), microglia activation, andcognitive dysfunction, targeting of the interleukin (IL)-1 pathway issufficient to reduced inflammation and ameliorate the disability.

Endotoxemia was induced in wild-type and IL-1R^(−/−) mice by intraperitoneal injection of E. Coli LPS (1 mg/kg). Markers of inflammationwere assessed both peripherally and centrally, and correlated tobehavioral outcome using trace fear conditioning.

Plasma increase in tumor necrosis factor-α (TNFα) peaked at 30 minutesafter LPS challenge. Up-regulation of IL-1β, IL-6 and HMGB-1 was morepersistent, with detectable levels up to day 3. A 15-fold increase inIL-6 and a 6.5-fold increase in IL-1β mRNA at 6 hours post intervention(p<0.001 respectively) was found in the hippocampus. Reactivemicrogliosis was observed both at days 1 and 3, and was associated withelevated HMGB-1 and impaired memory retention (p<0.005). Preemptiveadministration of IL-1 receptor antagonist (IL-1Ra) significantlyreduced plasma cytokines and hippocampal microgliosis and amelioratedcognitive dysfunction without affecting HMGB-1 levels. Similar resultswere observed in LPS-challenged mice lacking the IL-1 receptor to thewild type mice treated with IL-1Ra.

These data suggest that by blocking IL-1 signaling, the inflammatorycascade to LPS is attenuated, thereby reducing microglial activation andpreventing the behavioral abnormality. This amelioration appears to beindependent of HMGB-1 up regulation.

Materials and Methods Animals

All the experiments were conducted under the UK Home Office approvedlicense. Wild type C57BL/6 male mice, 12-14 weeks old, weighting 25-30 gwere used and housed in groups of five in a 12-h-12-h light dark cycle,with controlled temperature and humidity with free access to food andwater. IL-1R^(−/−) (kindly provided by Nancy Rothwell, University ofManchester [13]) were bred in-house on a C57BL/6 background andage-matched to wild type counterparts. Seven days of acclimatizationwere allowed before starting any experiment. All the animals werechecked on a daily basis and evidence of poor grooming, huddling,piloerection, weight loss, back arching and abnormal activity, wereexcluded in the experiments.

Treatment

LPS derived from Escherichia Coli endotoxin (0111:B4, InvivoGen, USA, 1mg/kg) was dissolved in normal saline and injected intraperitoneally.IL-1Ra (Amgen, Anakinra 100 mg/kg, Nederlands) was given subcutaneouslyimmediately before LPS administration. Dose response curve from LPS orIL-1Ra was obtained from our pilot studies to provoke or to suppressmoderate degree of microglia activation respectively. Control animalswere injected with equivalent volumes (0.1 ml) of saline. Mice from eachtreatment group were randomly assigned for assessment of either cytokineresponse or cognitive behavior, in order to obviate possible confoundingeffects of behavioral testing on inflammatory markers [14].

Plasma Cytokine Measurement

Blood was sampled transcardially after thoracotomy under terminalanesthesia 30 minutes, 2, 6, 12 hours and 1, 3, 7 days after experimentsin the different cohorts and centrifuged at 3,600 rpm for 7 minutes at4° C. Blood samples taken from animals without any interventions severedas controls. Plasma samples were stored at −20° C. for further analysis.Plasma cytokine and HMGB-1 were measured using commercially availableELISA kits from Biosource, CA and Shino-test Corporation, Japan,respectively. The sensitivities of the assays were <3 pg/ml for TNFα, <7pg/ml for IL-1β, <3 pg/ml for IL-6 and 1 ng/ml for HMGB-1.

Quantitative Real Time PCR (qPCR)

The hippocampus was rapidly extracted under a dissecting microscope,placed in RNAlater solution (Applied Biosystems, Ambion) and stored at4° C. Total RNA was extracted using RNeasy Kit (Qiagen) and quantified.The one-step qPCR was performed on a Rotor-Gene 6000 (Corbett LifeScience), using Assay-On-Demand premixed Taqmanprobe master mixes(Applied Biosystems). Each RNA sample was run in triplicate, andrelative gene expression was calculated using the comparative thresholdcycle ΔΔC_(t) and normalized to beta-actin (ACTB). Results are expressedas fold-increases relative to controls.

Immunohistochemistry (IHC)

Mice were euthanized and perfused transcardially with ice-coldheparinized 0.1M phosphate buffer solution (PBS) followed by 4%paraformaldehyde in 0.1M PBS at pH 7.4 (VWR International, UK). Thebrains were harvested and post-fixed in 4% paraformaldehyde in 0.1M PBSat 4° C. and cryoprotected in 0.1M PBS solutions containing 15% sucrosefor 24 hours (VWR International, UK) and then 30% sucrose for a further48 hours. Brain tissue was freeze-mounted in OCT embedding medium (VWRInternational, UK). The 25 μm thick coronal sections of the hippocampuswere cut sequentially in groups of 6 and mounted on Superfrost® plusslides (Menzel-Glaser, Germany). The rat anti-mouse monoclonal antibody,anti-CD11b (low endotoxin, clone M1/70.15) in the concentration of 1:200(Serotec, Oxford, UK) was used to label microglia. Visualization ofimmunoreactivity for CD11b was achieved using the avidin-biotintechnique (Vector Labs, Cambridge, UK) and a goat anti-rat secondaryantibody (Chemicon International, CA, USA) at a concentration of 1:200.A negative control omitting the primary antibody was performed in allexperiments. Immunohistochemical photomicrographs were obtained with anOlympus BX-60 microscope and captured with a Zeiss KS-300 colour 3CCDcamera. The assessment of staining, by an observer that was blinded tothe interventional group, was based upon a 4-point categorical scale[15].

Behavioral Measurement (Conditioning)

The behavioral study was conducted using a dedicated conditioningchamber (Med Associates Inc., USA). Mice were trained and tested onseparate days. LPS was injected within 30 minutes following training.The fear conditioning paradigm was used as previously described [16].Three days after training, mice were returned to the same chamber inwhich training occurred (context), and freezing behavior was recorded.Freezing was defined as lack of movement except that required forrespiration. Approximately 3 h later, freezing was recorded in a novelenvironment and in response to the cue (tone). The auditory cue was thenpresented for 3 min, and freezing scored again. Freezing scores for eachsubject were expressed as a percentage for each portion of the test.Memory for the context (contextual memory) for each subject was obtainedby subtracting the percent freezing in the novel environment from thatin the context.

Data Analysis

Statistical analyses were performed using GraphPadPrism version 5.0a(GraphPad Software, San Diego, Calif.). The results are expressed asmean ±SEM. Data were analyzed with analysis of variance followed byNewman-Keuls post hoc test wherever appropriate. For categorical data,non-parametric Kruskal-Wallis followed by Dunn's test was used. A p<0.05was considered to be statistical significance.

Results Endotoxin-Induced Cytokine Production is Modified by IL-1Ra andin IL-1R^(−/−)

To investigate the effects of inflammation on cognitive function wemeasured systemic and central cytokines after LPS administration. TNFαrelease occurred very rapidly and transiently; after 30 minutes it wassignificantly increased (104.18±7.36 pg/ml), peaking at 2 hours andreturning to normal at 6 hours post-injection (FIG. 9A; p<0.01, p<0.001vs control). LPS evoked a robust systemic response that induced furthercytokine release. Both IL-1β and IL-6 were significantly up regulatedfrom 2 hours. IL-1β increased 4-fold and plasma levels continued tosteadily increase until 24 hours (FIG. 9B; 73.49±5.42 pg/ml, p<0.001 vscontrol). IL-6 expression was highly elevated at 2 hours, decreasing at6 hours but still significantly detectable at 24 hours compared to naïveanimals (FIG. 9C; 134.37±8.43 pg/ml, p<0.01 vs control respectively).During this time, animals showed classic symptoms of sickness behavior(reduced motility, poor grooming, huddling, piloerection, back arching).Levels of HMGB-1 at 2, 6, and 12 hours post LPS were no different frombaseline levels; a 1.5-fold increase was observed from 24 hours afterLPS and remained elevated up to day 3 (FIG. 9D; 25.77±4.2 pg/ml, p<0.01,p<0.001 vs control). The systemic inflammatory response resolved afterday 3 and all cytokine levels returned to baseline by day 7.

To assess the central inflammatory response to LPS we measured levels ofIL-1β and IL-6 mRNA expression in the hippocampus. We noted a 6.5-foldincrease in IL-1β mRNA expression and a 15-fold increase in IL-6 in thehippocampus at 6 hours after LPS injection (FIG. 9E,F; p<0.001 vscontrol respectively). In both cases the increased trascription returnedto normal values by 24 hours. The increase in IL-1β both in plasma andin the hippocampus led us to investigate whether blocking the IL-1receptor could ameliorate the signs of LPS-associated cognitivedysfunction. A single preemptive dose of IL-1 Ra was able tosignificantly reduce plasma levels of IL-1β at 6 and 24 hours (FIG. 10A,32.7±5.45 pg/ml, 6.2±1.03 pg/ml, p<0.01 and p<0.001 vs LPSrespectively). Similarly, levels of IL-6 were also reduced at the sametime-points (FIG. 10B; 91.02±15.17 pg/ml, 14.05±2.34 pg/ml, p<0.001,p<0.001 vs LPS respectively). Interestingly, IL-1Ra treatment had noeffects on HMGB-1 levels, which maintained a similar pattern at thatseen after LPS injection in the absence of IL-1Ra (FIG. 10C).Corroboration of these data was achieved by injecting IL-1R^(−/−)animals with the same dose of LPS and measuring cytokine expression inplasma. At 24 hours, time characterized by increased cytokines and clearsickness behavior, levels of IL-1β and IL-6 were comparable to the wildtype mice that received IL-1Ra treatment (FIG. 10A,B; p<0.0001, p<0.001vs LPS respectively). However, upon measurement of HMGB-1 inIL-1R^(−/−), no differences were reported compared to either WT orIL-1Ra treated animals (FIG. 10C).

LPS-Induced Microglial Activation is Modified by IL-1Ra and Absent inIL-1R^(−/−)

The hippocampal trascriptome findings prompted interest for otherpossible markers of neuroinflammation. Microglia, the residentimmunocompetent cells of the CNS, were significantly up regulatedfollowing LPS injection. Minimal immunoreactivity was reported in naïveanimals in which cells maintained small cell bodies with thin and longramified pseudopodia (FIG. 11A). Resting microglia shifted to a“reactive profile” after LPS exposure, acquiring an amoeboid morphologywith hypertrophy of the cell body and retraction of the pseudopodia.Reactive microglia displayed morphological changes including increasedcell body dimensions, shortened and clumpy processes with higher levelsof CD11b immunoreactivity compared to naïve animals. Microglialactivation was reported at days 1 and 3 post exposure (FIG. 11B,C;p<0.01, p<0.05 vs control), returning to the baseline resting state byday 7. Pre-treatment with IL-1Ra effectively reduced the number ofreactive microglia at days 1 and 3 (FIG. 11E,F), with no changes at day7. In order to corroborate these findings, we repeated the experimentusing IL-1R^(−/−) animals and exposing them to LPS. No microglialactivation was noted in LPS treated IL-1R^(−/−) mice (FIG. 11G,H,I).

Hippocampal-Dependent Cognitive Dysfunction Following LPS is Amelioratedby IL-1 Blockade

To relate the inflammatory response to memory functioning, we used tracefear conditioning in which mice are trained to associate a tone with anoxious stimulation (foot shock). The brief gap between the tonetermination and the shock onset allows assessment of hippocampalintegrity [16]. The high level of freezing seen in the naïve animals isindicative of good learning and memory retention. Contextual fearresponse shows a reduced immobility (freezing) at day 3, revealing andhippocampal-dependent memory impairment (FIG. 12; p<0.005 vs naïvetrained). Pre-treatment with IL-1Ra significantly ameliorated thiscognitive dysfunction (FIG. 12; p<0.05 vs LPS).

Discussion

These data show that a sustained inflammatory challenge leads toneuroinflammation, microglial activation and hippocampal-mediatedcognitive dysfunction. By blocking the IL-1 receptor, the feed-forwardprocess that amplifies the inflammatory cascade is attenuated therebyreducing microglial activation and reversing the behavioral abnormalityafter endotoxemia.

Peripheral and Central Cytokines Contribute to the Inflammatory Milieuin Sickness Behavior

Cytokines play an important role in mediating the inflammatory responseafter infection or aseptic traumatic injury. The innate immunity israpidly triggered after LPS, primarily via activation of toll-likereceptor 4, TLR-4 [17]. Activation of TLR-4 induces a multitude ofpro-inflammatory cytokines via activation of transcription factors, NFκB[18]. This prompt response provides a favorable environment for thesynthesis and up regulation of both IL-1β and IL-6, which togethercontribute to the perpetuation of the inflammatory challenge. Also therapid increase in TNF-α following LPS, which is reported as presentalready after 30 minutes, promotes synthesis of other cytokines and theinitiation of the acute-phase response. Systemic cytokines, includingIL-1β, can bind receptors and translocate through the intact blood-brainbarrier (BBB) [19]. Neural afferents are known to be a fast and reliablepathway in the immune-to-brain signaling. Vagal-mediated signaling canrapidly induce brain cytokines and manifest the classic symptoms of theacute phase response, including neuroinflammation [20]. As we havereported a significant increase in both IL-1β and IL-6 mRNAtranscription at 6 hours in the hippocampus, the neuronal route may bethe likely pathway to trigger the early activation of these genes andthe initial changes in the CNS. Vagotomy was previously shown topartially attenuate sickness behavior both after LPS and IL-1βadministration [21], but not in the context of hippocampal-dependentcognitive dysfunction.

Reactive Microglia in the Hippocampus Interfere with Memory Processing

Within the brain, cytokines interact with microglia cells.Pro-inflammatory cytokines can directly interact with many of thepattern recognition receptors (PRRs) expressed on the surface of thesecells [22]. Upon activation, microglia exhibit discernible morphologicchanges and secrete cytokines, reactive oxygen species (ROS),excitotoxins (such as calcium and glutamate) and neurotoxins such asamyloid—β [23]. Activated microglia also inhibit neurogenesis in thehippocampus following endotoxemia, thereby exacerbating the extent ofinjury on memory processing [24]. To assess memory retention we usedtrace fear conditioning in which mice are trained to associate a footshock with a given environment or tone [25]. The extent to which ananimal freezes to a context is largely dependent on the hippocampus[26]. Hippocampal-dependent memory impairment was evident after 3 dayspost-LPS. Residual inflammation, primarily via reactive microglia, ispossibly associated with this second phase behavioral abnormality. Atthese time points, levels of HMGB-1 were also elevated and prompted usto further investigate the role of these factors in the development ofcognitive dysfunction.

Targeting IL-1 Ameliorates the Cognitive Abnormality by ReducingMicroglia but does not Affect HMGB1

IL-1β has a pivotal role in sustaining the neuroinflammatory responseand closely interacts with memory processing and long-term potentiation[27, 28]. Self-regulation and inhibition of IL-1β is normally achievedwith the neutralizing action of endogenous IL-1Ra, which directlycompetes for binding to the receptor [29, 30]. Transcription ofendogenous IL-1Ra would normally occur temporally delayed from thesynthesis of IL-1, thus following pharmacological intervention we aimedto block the receptor a priori impeding binding and limiting the damagemediated by the effector molecule. When the IL-1 receptor is disabled,either blocked pharmacologically (IL-1Ra) or by genetic intervention(IL-1R^(−/−)), the inflammatory response is not sustained as reflectedby lower cytokine release and microglia activation, thus amelioratingthe cognitive dysfunction as reported here. Treatment with IL-1Raprovides a significant improvement in cognitive dysfunction, confirmingthe crucial role of IL-1β in memory processes and behavior. Althoughthere was a temporal correlation between microglia activation andlate-release of HMGB-1, neither IL-1Ra nor IL-1R^(−/−) changed levels ofin this cytokine. This evidence supports the notion that blocking IL-1is sufficient to reduce the microglia activation and ameliorate thememory abnormality. Other receptors may be involved in sustaining thisinflammatory challenge; for example HMGB-1 has been shown to activateTLRs and receptor for advanced glycation end-products (RAGE) and it hasbeen reported as a key late pro-inflammatory mediator in sepsis, withconsiderable pathological potential [11, 31].

Some limitations of our study must be pointed out. Since IL-1Ra is ableto translocate directly into the brain [32], we are unable todiscriminate whether peripheral cytokines and/or de-novo production inthe CNS account for this cognitive dysfunction. Also, recently it hasbeen shown that peripheral monocytes can enter the brain causingsickness behavior. This process strongly relies on TNF-α signaling,especially in activating microglia and recruiting active monocytes intothe CNS [33]. In this study we cannot determine the nature of themicrogliosis, whether they are infiltrated macrophages that crossed theBBB or actual microglia.

CONCLUSION

The beneficial effects on cognition reported in this study by targetingIL-1, preemptively, are encouraging. However, it is not possible toextrapolate these benefits to the setting of cognitive dysfunction thataccompanies severe sepsis with multiple organ failure. In that clinicalscenario there are complex inflammatory responses that are difficult toreverse [34]. Clinical trials targeting IL-1 have been unconvincing inimproving mortality rate, especially in sepsis [35]. In this attempt tountangle the complexity of this condition, anti-IL-1 therapy appears tobe able to ameliorate the associated cognitive dysfunction,independently of other mechanisms. Inflammation clearly plays a pivotalrole in mediating physiological as well as behavioral changes afterLPS-exposure. Further studies are needed to ascertain whether selectivetargeting of other cytokine receptors can effectively prevent orameliorate both the degree and length of cognitive decline.

Key Messages

-   -   Neuroinflammation plays a pivotal role in mediating        physiological and behavioral changes after LPS    -   Up-regulation of microglia and HMGB-1 correlates in a temporal        fashion with the cognitive dysfunction    -   Blocking IL-1 does not affect HMGB-1 release, however it reduces        microglia activation reversing the behavioral abnormality    -   In the absence of IL-1, HMGB-1 is insufficient to sustain        hippocampal neuroinflammation and the attendant cognitive        dysfunction. Further studies are required to investigate the        potential benefit of anti-cytokine therapy in the ICU.

Abbreviations

High-mobility group box 1 (HMGB-1), interleukin (IL), lipopolysaccharide(LPS), toll-like receptor (TLR), tumor necrosis factor-α (TNFα)

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EXAMPLE 3 Anti-Cytokine Therapy for Postoperative Cognitive DysfunctionIntroduction

Postoperative cognitive dysfunction (POCD) is a well-describedcomplication that follows different surgical interventions, affectingprimarily the elderly (1). Etiology and mechanisms underlying thisphenomenon still remains elusive and not yet fully understood. Withsurgical operations already exceeding 230 million worldwide and theescalating surgical projections for the aging population, POCD will bean important complication with a significant burden on patients andhealth care management (2-4).

POCD is associated with poor short-term and long-term outcomes such asincreased risk of mortality and comorbidities, including the possibilityof permanent dementia and further neurodegeneration (5, 6). Multiplefactors (patient-related, perioperative care and surgery) are thought tomodify the risk of developing POCD; to date clinical studies have notrevealed the mechanisms for cognitive disturbances (7). Exploitation ofthe clinical knowledge into animal models provides novel insights intothe risk factors and mechanisms in this condition (8, 9). We postulatedthat targeting tumor necrosis factor-α (TNFα) can block the perpetuationof the inflammatory challenge and ameliorate the cognitive dysfunction.

Methods Animals

All the experiments were conducted under the UK Home Office approvedlicense. Wild type C57BL/6 male mice (Harlan, UK) 12-14 weeks old wereused. MyD88^(−/−) (10) and TLR4^(−/−) were bred in house on a C57BL/6background and age-matched to wild type counterparts. All the animalswere checked on a daily basis and if they evidenced poor grooming,huddling, piloerection, weight loss, back arching and abnormal activity,they were eliminated from further consideration.

Surgery

Mice were subjected to an open tibial fracture, stripped of periosteumwith intramedullary fixation, under aseptic conditions and generalanaesthesia with inhaled isoflurane (MAC 1.5) and analgesia withbuprenorphine as previously described (11). Untreated animals served asnaïve controls. TNF neutralizing antibody (clone TN3, Sigma, UK, 100 μgin 0.1 ml/volume) was dissolved in normal saline and injected 18 hoursbefore surgery. LPS derived from Escherichia Coli endotoxin (0111:B4,InvivoGen, USA, 1 mg/kg) was dissolved in normal saline and injectedintraperitoneally to serve as positive control (data not shown).

Cytokine Analyses

Systemic and hippocampal IL-1β was measured by ELISA (Bender Medsystem,CA; 1.2 pg/ml of sensitivity), as previously described (12).

Immunohistochemistry

Fixed brains were collected for immunohistochemical DAB staining formicroglia activation using CD11b and scored as previously described(13).

Behavioral Measurement (Conditioning)

The behavioral study was conducted using a dedicated conditioningchamber (Med Associates Inc., USA). Mice were trained and tested onseparate days. The fear conditioning paradigm was used as previouslydescribed (14). Three days after training, mice were returned to thesame chamber in which training occurred (context), and freezing behaviorwas recorded. Freezing was defined as lack of movement except thatrequired for respiration. Freezing scores for each subject wereexpressed as a percentage for each portion of the test. Memory for thecontext (contextual memory) for each subject was obtained by subtractingthe percent freezing in the novel environment from that in the context.

Data Analysis Statistical analyses were performed using GraphPadPrism(GraphPad Software, San Diego, Calif.). The results are expressed asmean ±SEM. Data were analyzed with analysis of variance followed byNewman-Keuls post hoc test wherever appropriate. For categorical data,non-parametric Kruskal-Wallis followed by Dunn's test was used. A p<0.05was considered to be statistical significance.

Results

To investigate the effects of inflammation on surgery-induced cognitiveabnormalities we targeted TNFα as the putative initiator of thisprocess. A positive trend in systemic TNFα, from 30 to 60 minutes, wasobserved following tibial surgery (see FIG. 13).

Preemptive administration of anti-TNFα effectively reduced the amount ofsystemic IL-1β both at 6 hours and 24 hours following tibia surgery(p<0.01 and p<0.001 vs surgery only respectively). To corroborate thefindings and ascertain the specificity of TNF, we delayed the injectionof the antibody and levels of IL-1β remained unaffected (see FIG. 14A).To better understand the effects of the TNFα blockade on other cytokineswe also measured levels of IL-6 as downstream products from IL-1receptor. Prophylaxis with anti-TNFα reduced systemic levels of IL-6both at 6 and 24 hours (p<0.01 and p<0.05 vs surgery only respectively).If the TNFα blockade was delayed, no effects were observed on IL-6similarly to IL-1 (see FIG. 14B). In order to correlate the systemicchanges with central markers of inflammation, we measured levels ofIL-1β and assessed microglia activation in the hippocampus. Prophylaxiswith anti-TNF significantly reduced the levels of hippocampal IL-1βcompared to untreated animals (see FIG. 14C, p<0.01). Microglia, theresident immunocompetent cells of the CNS, shifted their state to a“reactive profile” after surgery, acquiring an amoeboid morphology withhypertrophy of the cell body and retraction of the pseudopodia (see FIG.14E). Minimal immunoreactivity was reported in naïve animals in whichcells maintained small cell bodies with thin and long ramifiedpseudopodia (see FIG. 14E). Treatment with anti-TNF reduced the amountof microgliosis seen after surgery (see FIG. 14F, G; p<0.01 vs surgery).To relate the inflammatory response to memory functioning, we used tracefear conditioning (TFC) in which mice are trained to associate a tonewith a noxious stimulation. The brief gap between the tone terminationand the shock onset allows assessment of hippocampal integrity (15). Thehigh level of freezing seen in the naïve animals is indicative of goodlearning and memory retention. No differences in freezing time werereported between groups during training (data not shown). Contextualfear response however shows a reduced immobility (freezing) atpostoperative day 3, revealing hippocampal-dependent memory impairment(see FIG. 14H; p<0.05 vs naïve trained). Pre-treatment with anti-TNFsignificantly ameliorated this cognitive dysfunction (see FIG. 14H;p<0.05 vs surgery). Also, administration of anti-TNF immediately aftersurgery still under effects of general anesthesia, provided a similaramelioration (data not shown).

The effects of anti-TNFα prophylaxis, in particular on IL-1β, providedfurther insights into the possible role of IL-1 and non-TNFα signallingin POCD-associated behavior. Reduction of systemic inflammation both at6 and 24 hours was observed in MyD88^(−/−) (see FIG. 15A, B), reachingsimilar values after prophylaxis in WT animals. In order to correlatethe systemic changes with neuroinflammation and eventual behavioralabnormality, we assessed hippocampal IL-1β and microglia activation. Nosigns of neuroinflammation were reported in MyD88^(−/−) followingsurgery (see FIG. 15C, F). In order to understand the role ofMyD88^(−/−) in POCD-associated behaviour we used TFC to assess memoryretention following surgery. Contextual retrieval task revealed nosignificant changes in freezing behavior comparing naïve MyD88^(−/−) toanimals receiving tibia surgery (see FIG. 15G). Despite the significantreduction in systemic IL-1β both following anti-TNFα prophylaxis andusing MyD88^(−/−), the IL-1 response was not obliterated. This led us tofurther investigate whether a putative synergistic interaction betweenTNFα and IL-1β could account for sustaining the response. WhenMyD88^(−/−) were treated with preemptive anti-TNFα, the response toIL-1β and IL-6 was completely eliminated (see FIG. 15H, I; p<0.001,p<0.01 vs surgery respectively).

To further understand the involvement of MyD88-dependent signaling inPOCD, we investigated the inflammatory response in TLR4^(−/−). Only asubtle reduction in plasma levels of IL-1β and IL-6 was observed at 6hours following surgery, but by 24 hours pro-inflammatory cytokines wereunchanged from WT. The systemic response was fully abrogated followingtreatment with anti-TNFα, similarly to what observed after prophylaxisin MyD88^(−/−) (see FIG. 16A, B). Furthermore, administration ofanti-TNFα resolved the neuroinflammatory signs present in TLR4 ^(−/−)following surgery, including hippocampal IL-1β (see FIG. 16C, p<0.01 vsab) and microglial activation (see FIG. 16G, p<0.01 vs naïve and ab). Tocorroborate the importance of this inflammatory challenge withPOCD-associated behavior, TFC revealed a clear hippocampal impairmentfollowing surgery in TLR4, similarly to WT (see FIG. 16H, p<0.05 vsnaive).

Discussion

These data demonstrate that anti-TNFα monoclonal antibody reduces theinflammatory burden following surgery, through the elaboration of IL-1β.The importance of IL-1β in POCD can be seen from Example 1 above. Hereinwe demonstrate that preemptive targeting of TNFα as an early markerduring post-surgical inflammation suppresses generation of IL-1,strengthening the role of cytokines in the etiology of POCD. In order todefine the origin of the IL-1 response, we also explored MyD88- andTL4-dependent signaling.

Given the limited penetration of the antibody into the brain, our datasuggest a pivotal role for the initial peripheral response in thedevelopment of neuroinflammation and POCD-associated behaviouralchanges. Reduction of IL-1β through TNFα improves behavioralperformance, suggesting a key role of inflammation in the development ofbehavioral abnormalities and further proving the determinant role ofIL-1 in memory processes (16, 17).

Due to the recent discoveries of independent MyD88 pathways in the IL-1signaling (18), we investigated the response following surgery inanimals lacking expression of this key adaptor molecule. MyD88^(−/−)showed a reduction, albeit not complete, in IL-1β. Dampening of theinflammatory challenge appears to be sufficient to provide benefitPOCD-associated behavior following surgery in MyD88. This suggests athreshold effect in which a certain level of IL-1 is required to triggerenough neuroinflammation to impact on memory functioning resulting inthe behavioral changes. Combination of anti-TNFα prophylaxis toMyD88^(−/−) was able to completely abrogate the response to IL-1β. Theresults from the TFC also suggest that the elimination of eithercytokines will diminish cognitive dysfunction following surgical trauma(i.e. threshold effect).

The demonstration of the importance of MyD88 signaling in POCD yieldedto further investigate whether targeting of specific receptors couldameliorate POCD symptoms. Due to the findings on the involvement of TLR4during sterile inflammation and trauma (fracture) (19), we sought tolook at the inflammatory response in TLR4^(−/−). Both IL-1β response andcognitive dysfunction after surgical stimulation was not eliminated.However, anti-TNFα prophylaxis was able to reduce the inflammatorychallenge in TLR4^(−/−), further proving the key role of TNFα in thegeneration of IL-1β through a MyD88 independent pathway followingsurgery.

Herein we provide evidence for IL-1β working through MyD88 mechanismbut, in addition, targeting of TNFα significantly ameliorates thecognitive dysfunction after surgery by reducing IL-1β. Therapy with TNFinhibitors already offers beneficial effects in other settings such asrheumatoid arthritis (20). Following a single preemptive administrationwe reported no evidences of infection or sickness behavior in thisstudy, thus overcoming some of the limitations of these agents infacilitating postoperative complications, in particular infections.

REFERENCES

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EXAMPLE 4 The Interactions Between Postoperative Infection, Surgery, andInflammation in Post-Operative Cognitive Dysfunction BACKGROUND

Recovery from surgery may be complicated by postoperative cognitivedysfunction (POCD), especially in high-risk patients [1]. Postoperativecomplications, for example infection, have been associated with higherincidence of POCD although the mechanisms governing the interaction inthe pathogenesis of POCD are not known [2]. Recently, neuroinflammationhas been correlated with cognitive decline [3,4]. In this study wesought to understand the effect of postoperative lipopolysaccharide(LPS) on inflammation and POCD-associated behavior after orthopedicsurgery.

Methods

Adult C56BL/6J male mice were randomly assigned into groups thatwere: 1) untreated (naïve) animals; 2) tibial fracture under GA andanalgesia; 3) 24 h following tibial surgery, i.p. injection of LPS (1mg/kg) 4) LPS injection only. Separate cohorts of mice per group wereassessed for inflammatory markers (plasma cytokines and microglialactivation), or hippocampal-dependent memory using trace fearconditioning (TFC).

Results

TFC assessment at three days after surgery and LPS revealed asignificant reduction in freezing behavior compared to both naïvelittermates and animals undergoing surgery only, without complication(p<0.05 vs surgery). Up-regulation of systemic cytokines is usuallyself-limited to the initial 24 h; however, postsurgical administrationof LPS up-regulated plasma levels of IL-1 for 72 h followingintervention (p<0.001 vs control). In the hippocampus we reported higherdegree of reactive microgliosis (CD11b) in animals treated with LPScompared to surgery or naïve; microgliosis was reported up topostoperative day 7 in the postsurgical LPS group (p<0.05).

Conclusion

Cytokines are pivotal mediators in triggering and sustaining cognitivedysfunctions following aseptic inflammation following tibial fracture.Supervention of infection following aseptic trauma exaggeratesinflammation and thereby exacerbates POCD. The individual contributionsand their convergence on the inflammatory pathways will help definepotential targets for intervention.

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1. A method for preventing or reducing cognitive decline in a patientfollowing a planned inflammatory trigger in said patient, the methodcomprising administering a therapeutically effective amount of a TumourNecrosis Factor alpha (TNFα) antagonist to said patient.
 2. The use of atherapeutically effective amount of a Tumour Necrosis Factor alpha(TNFα) antagonist in the manufacture of a medicament for use inpreventing or reducing cognitive decline in a patient following aplanned inflammatory trigger in said patient.
 3. An agent for use inpreventing or reducing cognitive decline in a patient following aplanned inflammatory trigger in said patient, wherein the agentcomprises a therapeutically effective amount of a Tumour Necrosis Factoralpha (TNFα) antagonist. 4.-61. (canceled)